Method of enhancing an immune response

ABSTRACT

A method of enhancing an immune response to an antigen is provided. The method involves augmenting the level of a TAP molecule in a target cell bearing the antigen. Preferably, the TAP molecules enhanced by administering a nucleic acid sequence encoding a TAP-1 and/or TAP-2 molecule. The method is useful in treating infectious diseases and cancer.

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 08/817,731 filed Jul. 21, 1997 (now allowed), whichis a national phase entry application of PCT/CA95/00544 filed Sep. 22,1995, which claims priority from U.S. patent application Ser. No.08/311,442 filed Sep. 23, 1994, now abandoned.

FIELD OF THE INVENTION

The invention relates generally to a method of enhancing an immuneresponse to an antigen by augmenting the level of a TAP molecule in atarget cell bearing the antigen.

BACKGROUND OF THE INVENTION

The cytotoxic T lymphocyte (CTL) response is a major component of theimmune system, active in immune surveillance and destruction of infectedor malignant cells and invading organisms expressing foreign antigens ontheir surface. The ligand of the antigen specific T cell receptor is acomplex made up of a peptide fragment of an antigen bound to majorhistocompatibility complex (MHC) molecules. In particular, cytotoxic Tlymphocytes recognise peptide bound to MHC Class 1 molecules.

MHC class 1 molecules are normally expressed at the cell surface asternary complexes formed by a heavy chain of 46 kD, a light chain called82-microglobulin (β₂M) of 12 Kd and a peptide composed of 8-10amino-acids Ivan Bleek, G. M. and S. G. Nathenson, Nature 348:213, 1990;Zhang, W. et al., Proc. Natl. Acad. Sci. USA 89:8403, 1992; Matsumura,M. et al., Science 257:927, 1992; and Latron. F., et al., Science257:964, 1992). Formation of the ternary complex is thought to involvetransport into the lumen of the endoplasmic reticulum (ER) of peptidesgenerated by protein degradation in the cytoplasm (Nuchtern. J. G. etal., Nature 339:223, 1989; Yewdell, J. W. and J. R. Bennink, Science244:1072, 1989; and Cox. J. H. et al., Science 247:715, 1990). The studyof mutant cell lines selected for their low expression of MHC class 1molecules at the cell surface has provided insights into the molecularevents required for antigen processing. These studies have allowed theidentification of two genes located in the MHC region which encodeproteins of the ATP binding cassette (ABC) family. These genes, calledTAP-1 and TAP-2, have been implicated in transport of peptides from thecytoplasm to the lumen of the ER (Deverson, E. V. et a). Nature 348:738,1990; Trowsdale, J. et al., Nature 348:741, 1990; Spies, T. et al.,Nature 348:744, 1990; Monaco, J. J. et al., Science 250:1723, 1990;.Spies, T. and R. DeMars, Nature 351:323, 1991; Bahram, S. et al., Proc.Natl. Acad. Sci. USA 88:10094, 1991; Spies, T. et al., Nature 355:644,1992; Kelly, A. et al., Nature 355:641, 1992; Powis, S. H. et al., Proc.Natl. Acad. Sci. USA 89:1463, 1992; and Colonna. M. et al., Proc. Natl.Acad. Sci. USA 89:3932, 1992). Two other MHC linked genes. LMP-2 and -7(Monaco, J. J. and McDevitt, 1982, Proc. Natl. Acad. Sci. USA 79:3001),are components of the proteasome, a cytoplasmic multicatalytic proteasecomplex, which is likely responsible for some aspects of proteindegradation for antigen processing (Ortiz-Navarette, V. et al., Nature353:662, 1991; Brown, M. G. et al., Nature 353:355, 1991; Glynne, R. etal., Nature 353:357, 1991; Martinez, C. K. and J. J. Monaco, Nature353:664, 1991; Kelly, A. et al., Nature 353:667, 1991; Yang, Y., et al.,Proc. Natl. Acad. Sci. USA 89:4928, 1992; Goldberg, A. L. and K. L.Rock, Nature 357:3751 1992).

The mouse mutant lymphoma cell line RMA-S expresses low levels of classI molecules at the cell surface compared to the wild type RMA cells(Ljunggren, H.-G. et al., J. Immunol. 142:2911, 1989; and Townsend. A etal., Nature 340:443, 1989). Influenza virus infected RMA-S cells presentinfluenza peptides in the context of D^(b) molecules inefficiently andare only weakly recognized by specific CTL (Townsend, A et al., Nature340:443, 1989). Transfection with the putative transporter gene, TAP-2,complements this deficiency (Powis. S. J. et al., Nature 354:528, 1991;and Attaya. M. et al., Nature 355:647, 1992). The endogenous TAP-2 geneof RMA-S cells was shown to contain a point mutation which introduces astop translation codon resulting in an incomplete and defective TAP-2protein (Yang. Y. et al., J. Biol. Chem. 267:11669, 1992). Despite thedefective TAP-2 protein in RMA-S cells, antigenic peptides fromvesicular stomatitis virus (VSV) bypass the defect and are presented tospecific CTL by K^(b) molecules in RMA-S cells (Esquivel. F., et al., J.Exp. Ned. 175:163, 1992; and Hosken. N. A. and M. J. Bevan, J. Exp. Med.175:719, 1992). The VSV-nucleocapsid (N) peptide, VSV-N 52-59, has beenshown to be the major peptide presented by K^(b) molecules on VSVinfected cells (van Bleek, G. M. and S. G. Nathenson, Nature 348:213,1990). The presence of the wild-type TAP-1 protein in RMA-S cells may besufficient for translocation of the VSV-N 52-59 peptide to the ER lumen(Powis. S. J. et al., Nature 354:528, 1991; Attaya, M. et al., Nature355:647, 1992; and Yang. Y. et al., J. Biol. Chem. 267:11669, 1992).Alternatively, the VSV-N 52-59 peptide may not need a functionaltransporter for transport into the lumen of the ER. Expression ofminigene-encoded viral peptide epitopes in T2 cells (Zweerink. H. J. etal., J. Immunol. 150:1763, 1993) and in-vitro translation andtranslocation using microsomes from T2 cells (Lévy, F. et al., Cell67:265, 1991) support this contention.

A separate class of antigen processing variants are those in which theassembly and the surface expression of MHC class I molecules areentirely inducible by IFN-γ (Klar, D. and G. J. Hc1immerling, EMBO J.8:475, 1989). For example in the small lung carcinoma cell line. CMT.64,recognition by influenza virus specific CTL does not take place unlessinduced with IFN-γ (Sibille, C. et al., Eur. J. Immunol. 22:433, 1992).The very low amount of all proteasome components present in uninducedCMT.64 cells is presumed to be responsible for their phenotype(Ortiz-Navarette. V et al., Nature 353:662, 1991). Exogenous influenzapeptides can bind to D^(b) molecules on CMT.64 cells and complementrecognition by influenza specific CTL (Sibille, C. et al., Eur. J.Immunol. 22:433, 1992). In addition, it has been found that the β₂m andthe VSV-N 52-59 peptides added exogenously to these cells complementrecognition by VSV specific CTL restricted to K^(b) (Jefferies W. A. etal., 1993. J. Immunol. 151:2974). The amount of β2m and of heavy chainssynthesized in these cells may limit the amount of MHC class Iexpression on the cell surface (Jefferies et al, supra, 1993). Adysfunction of the putative peptide transporters and/or in thegeneration of the peptide may be responsible for the CMT.64 phenotypewhich may represent a mechanism to downregulate MHC class I expression,a feature common to many carcinomas.

Restifo. N. R. et al. (J. Exp. Ned. 177:265-272, 1993) studied theantigen processing efficiency of 26 different human tumor lines using arecombinant vaccinia virus (VV) to transiently express the K^(d)molecule. Three cell lines, all human small cell lung carcinoma,consistently failed to process endogenously synthesized proteins forpresentation to K^(d)-restricted, Vac-specific T cells. Pulse-chaseexperiments showed that MHC class 1 molecules were not transported bythe cell lines from the endoplasmic reticulum (ER) to the cell surface.Northern blot analysis of the cells revealed low to nondetectable levelsof mRNAs for MHC-encoded proteasome components LMP7 and LMP-2 as well asthe putative peptide transporters TAP-1 and TAP-2.

There is a need in the art for methods to augment or enhance an immuneresponse to various targets including virally infected and malignantcells.

SUMMARY OF THE INVENTION

The present inventors have shown that augmenting either TAP-1 or TAP-2expression on either virally infected or tumor target cells enhances theimmunogenicity of the target cells as demonstrated by an enhancedcytotoxic T lymphocyte response.

Accordingly, the present invention provides a method of enhancing animmune response to antigen by administering an effective amount of anagent that can augment the level of a TAP molecule in a target cellbearing the antigen to a cell or animal in need thereof.

In one embodiment, the level of the TAP molecule is augmenting byadministering a nucleic acid sequence encoding the TAP molecule to atarget cell bearing the antigen.

The target cell can be any cell to which one wishes to generate animmune response such as a virally infected cell or a cancer cell. Thetarget cell can also be normal or non-infected cell, for example whenthe method is used as a vaccine or in a prophylactic protocol togenerate an immune response to a particular antigen.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1A depicts histograms showing CTL recognition of VSV infected RMAand RMA-S IFN-γ induced (+);

FIG. 1B depicts histograms showing CTL recognition of VSV infectedCMT.64 cells (abbreviated: C) IFN-γ induced (+) or uninduced cells;

FIG. 2 is an autoradiogram showing the amount of β₂m synthesized in RMA.RMA-S and CMT.64. IFN-γ induced (+) or uninduced (−) cells;

FIG. 3 is a histogram showing the effect of β₂M on the CTL responseagainst CMT.64 cells superinfected with vaccinia virus (VV) andVaccinia-β₂m recombinant (VV—β₂m), Vaccinia and VSV (V-VSV), rVV-VSV orVaccinia-β₂m, and VSV (Vb2-VSV), the insert shows the level of β₂msynthesized after immunoprecipitation with the anti-hβ₂m rabbit serum;

FIG. 4 depicts autoradiograms showing the intracellular transport of MHCclass I molecules, D^(b) and K^(b);

FIG. 5 depicts autoradiograms showing the intracellular transport offree and assembled forms of K^(b) molecules in uninduced andIFN-γ-induced CMT.64 cells;

FIG. 6 is a histogram showing VSV-N 52-59 peptide dose response in CTLrecognition for CMT.64 cells (CMT), CMT.64+IFN-γ (CMT+IFN-γ) and RMA-Scells (RMA-S);

FIG. 7 shows Northern blot analysis of cytoplasmic RNA from RMA, RMA-S,CMT.64 IFN-γ induced and uninduced cells;

FIG. 8 shows two dimensional gel analysis of proteasome components fromRMA. RMA-S and CMT.64 IFN-γ induced or uninduced cells;

FIG. 9 shows CMT 64 (CMT), and CMT 64 transfected with TAP-1 (CMT TAP 1)infected with or without VSV for 8 hr at MOI of 5, or treated withN52-59 peptide for 2 hr at 500 μM (50% dose response);

FIG. 10 shows TAP gene expression profiles of CMT.64 and CMT64/rl-4;

FIG. 11 shows flow cytometric analysis demonstrating that TAP-1expression is sufficient to increase levels of K^(b) and D^(b) at thecell surface of CMT.64 cells;

FIG. 12 shows pulse-chase analysis of K^(b) and D^(b) molecules fromCMT.64 and TAP-1 transfected CMT.64 cells (CMT64/rl-4);

FIG. 13 is a histogram showing that TAP-1 transfected CMT.64 cells(CMT-rl.4) efficiently present antigen to VSV specific CTL;

FIG. 14 is a histogram showing that TAP-1 and TAP-2 transfected CMT.64cells efficiently present antigen to VSV specific CTL;

FIG. 15 is a histogram showing that various TAP-1 transfected clonesefficiently present antigen to VSV specific CTL;

FIG. 16 is a histogram showing that RMA cells and CMT.64 cells treatedwith IFN-γ are recognized efficiently after influenza infection andCMT.64 cells transfected with the rat TAP-1 gene are not recognized;

FIG. 17 is a histogram showing that RMA cells and CMT.64 cells treatedwith IFN-γ are recognized efficiently after influenza infection and CMT.64 cells transfected with the rat TAP-1 gene are not recognized;

FIG. 18 is a histogram showing that RMA cells and CMT.64 cells treatedwith IFN-γ, CMT.64 cells transfected with both rat TAP genes arerecognized after influenza virus infection;

FIG. 19 is a histogram showing that HSV infected cells are recognized byspecific CTL independently of the expression of the rat TAP-1 and/orTAP-2 transporter genes;

FIG. 20 is a graph showing survival of C57B1/6 and Balb/C mice injectedwith 5×10⁵ CMT.64 or CMT.12.12 cells;

FIGS. 21A and B are graphs showing the immune response to varying VV-NPdosage. Splenocytes from immunized C57Bl/6 mice were tested for theirability to recognize VSV infected targets in a 4 hour ⁵¹Cr release CTLassay. The mice were injected ip.with either VSV (3×10⁷ TCID50), orVV-NP at 10³ (3), 10⁴ (4), 10⁵ (5), 10⁵ (6) pfu, or VV-pJS5 (10⁶ pfu),or PBS. The splenocytes were tested for their ability to recognizeeither A) RMA targets infected with VSV (MOI=10 for 8 hours) or B)uninfected RMA targets;

FIGS. 22A and B are graphs showing the specificity of splenocytes fromVV-NP immunized mice. Splenocytes from immunized { }57B|/6 mice weretested for their ability to recognize targets in a 4 hour ⁵¹Cr releaseassay. The mice were injected ip. with either VSV (3×10⁷ TCID50), orVV-NP (10⁶ pfu), or VV-pJS5 (10⁶ pfu), or PBS. They were tested fortheir ability to recognize either A) RMA targets pulsed for one hourwith VSV N peptide or B) RMA targets infected with VV-pJS5 (MOI=10 for 8hours);

FIG. 23 is a graph showing the effect of TAP on VV—NP immunization. (A)Splenocytes from C57Bl/6 mice immunized ip. with VV-NP (106 pfu) with orwithout VV-hTAP (5×10⁶ pfu) were tested for their ability to recognizeVSV infected RMA targets (MOI=10 for 9 hours) in a 4 hour ⁵¹Cr releaseassay. (B) Frequencies of VSV specific CTL in splenocytes from miceinjected with VV—NP alone or with VV-hTAP12 were determined in alimiting dilution analysis;

FIG. 24 is a graph showing the effect of TAP on a low dose immunizationSplenocytes from C57Bl/6 mice were tested against VSV NP (1 mM) pulsedRMA targets in a standard 4 hour ⁵¹Cr release assay. The mice wereimmunized with VSV (2.7×10³ TCID₅₀) alone or with VV-hTAP12 (1.35×10⁴pfu);

FIG. 25 is a graph showing the VSV-specific primary CTL generation inVSV-infected mice is viral dose-dependent. The immunized splenocytesderived from the mice injected with the indicated VSV TCID50 doses weretested of their cytotoxic activity by using VSV-Np peptide-pulsed RMAcells as target. A standard 4-h 51Cr release assay was performed;

FIG. 26 is a graph showing that TAP heterodimer enhances theVSV-specific primary CTL response. The immunized splenocytes derivedfrom the mice injected either VSV alone or VSV plus VV carrying with orwithout humen TAP 1 and 2 genes were tested for their cytotoxic activityby using VSV-Np peptide-pulsed RMA cells as target. A standard 4-h ⁵¹Crrelease assay was performed. Each legend is shown as following,VV-PJS-5+VSV low—3×10⁴ (PFU) VV alone+3.6×10⁴ (TCID50) VSV, VV-TAP1,2

+VSV low—3×10⁴ (PFU) VV carrying human TAP 1 and 2+3.6×10⁴ (TCID50) VSV,VSV low 3.6×10⁴ (TCID50) VSV and VSV high—1.5×10⁷ (TCID50);

FIG. 27 is a graph showing VSV-Np epitope specific CTLp frequency wastremendously enhanced by introducing TAP 1 and 2 genes intoVSV-immunized mouse. The immunized splenocytes from the mice injectedwith either VV-TAP1,2+VSV low or VV-PJS-5+VSV low with viral dosesindicated in FIG. 26 legend were analyzed for VSV-Np epitope specificCTLp frequency. A limiting dilution analysis was performed by usingVSV-Np peptide-pulsed RMA cells as target;

FIG. 28 is a bar graph showing murine splenocytes express introducedhuman TAP protein and efficiently transport a peptide-library. 3×104 or3×105 PFU of either VV-PJS-5 or VV-TAP1,2 were injected i.p. into mice.After injection one day, the splenocytes were removed and detected forhuman TAP1 expression and TAP transport activities. The naive (Normal)and TAP−/− mice splenocytes are used as controls. A. The immunoblotinganalysis was performed to detect human TAP 1 expression. B. TAP functionwas tested by a peptide transport assay using a ¹²⁵I-labeled peptidelibrary as reporter; and

FIG. 29 is a graph showing TAP heterodimer enhances the Sandaivirus-specific primary CTL response. The immunized splenocytes derivedfrom the mice injected either Sandai virus alone or Sandai virus plus Wcarrying with or without humen TAP 1 and 2 genes were tested for theircytotoxic activity by using 5 μM Sendai-Np (324-332) peptide-pulsed RMAcells as target. A standard 4-h 51Cr release assay was performed. Eachlegend is shown as following, VV-PJS-5+Sandai low 3×10⁴ (PFU) VValone+1.58×10⁵ (CEID50) VSV, VV-TAP1,2+VSV low 3×104 (PFU) VV carryinghuman TAP 1 and 2+1.58×10⁵ (CEID50) Sandai virus, Sandai low 1.58×10⁵(CEID50) Sandai virus and Sandai high 1.58×10⁷ (CEID50) Sandai virus.

FIG. 30 is a FACS analysis showing reconstitution of the MHC Class Ilevel on the surface of mouse prostate cells using Interferon-γ or TAP1gene therapy. (A) 148-1 LMD metastatic cancer cells untransfected(green) or transfected with TAP1 (red) were examined by FACS for thelevel of expression of MHC Class I by staining with the Y3 anti-H2 Kbantibody. (B) 148-1 PA untreated (green) or treated with 400 U/mlInterferon-γ (red) were examined by FACS for the level of expression ofMHC Class I by staining with the Y3 anti-H2 Kb antibody. Purpleindicates the staining control.

FIG. 31 is a FACS analysis of B16 cells transfected with TAP1 measuresan increase in antigen loaded MHC class I on cell surface overuntransfected and vector alone transfected cells. Increases in theexpression of both a) K^(b) and b) D^(b) forms of MHC class I. RMA cellsare used as a positive control. The substitution of PBS for the primaryantibody for B16 serves as negative control.

FIG. 32 is an agarose gel electrophoresis of RT-PCR products generatedwith primers specific for rTAP 1(a), and rTAP 2 (b) is used to confirmthe Transfection of B16 F10 cells. β-actin was used to control forreverse transcription and template loading of the PCR.

FIG. 33 is a bar graph showing TAP1 expression enhanced the presentationof the tumour associated antigen. TRP-2, by B16 F10 cells. The TRP-2antigen presentation capability of B16 F10 cells alone was compared withthat of B16 F10 cells transfected with TAP1. In ⁵¹Cr release assays, theeffectors were obtained by injecting mice with γ-irradiated RMA-8 cellspulsed with the TRP-2 peptide epitope. The spleens of the injected micewere taken 5 days later. The splenocytes were then cultured in completeRPMI medium and restimulated by the TRP-2 peptide epitope for 5 days.These restimulated splenocytes were tested to be specific for TRP-2presented by H2-K^(b) molecules (data not shown). Standard 4-hour ⁵¹Crrelease assays were performed using the restimulated splenocytes aseffectors and B16 cells or B16 cells transfected with TAP1 as targets.B16 cells pulsed with the peptide epitope were used as the positivecontrol. The results showed that in TAP1 expressing B16 F10 cells thepresentation of TRP-2 was augmented significantly. TAP1 enhanced thepresentation of an endogenous tumour associated antigen by B16 F10tumour cells.

FIG. 34 is a graph showing increased specific killing of B16 cellstransfected with TAP1 compared with B16 F10 cells not transfected withTAP1. Cytoxic T lymphocytes are specific for the melanoma-associatedantigen. Untransfected B16 cells pulsed with TRP-2 antigen are used as apositive control.

FIG. 35 is a bar graph showing subcutaneous injection of a vacciniavector containing TAP1 inhibits significantly (one tail Student'st-test, p<0.5) the growth of established B16 melanoma tumors 3 fold whencompared to tumor growth in mice treated with subcutaneous injections ofvaccinia vector alone. The experiment used mice syngeneic with B16 cellline.

FIG. 36 shows the increase of CMT.64 antigenicity by transfection withTAP1 but not TAP2. A. rTAP1 and rTap2 expression of the transfectantsare shown by Western blots. B. Target cells pulsed with \/8V-Np peptidewith indicated concentrations for 1 hour following performingcytotoxicity assay. 50:1 E:T ratio is shown. C. Target cells wereinfected with 1:10 m.o.i. VSV overnight prior to examination of antigenpresentation capacity with a ⁵¹Cr-release assay. 100:1 ratio is shown.

FIG. 37 shows the control of in vivo-tumor-growth and improvement ofmice survival by introducing rTAP heterodimer or rTap1 but not rTap2into CMT.64 tumor cell line. CMT.64, or its tranfectants were injectedip. Into syngeneic mice. A after one month, one representative mousefrom each group was sacrificed and the tumor-growth pattern wasexamined. I—CMT.neo, II—CMT.1-10, III—CMT 2-10 and IV—CMT.12-21. Arrowsindicate tumors. B. The time of morbidity in mice survival experimentswas recorded for each group. Statistical analysis yields P-value,comparing survival rates of CMT-64-bearying mice (top) orCMT.neo-bearing mice (bottom). C. The specificity of splenocytes from amouse injected with CMT.neo (left-panel) or CMT 1-4 (right panel) wasdetermined in a CTL assay against the targets CMT.neo and CMT1-4.

FIG. 38 shows the percentage of tumor-infiltrating lymphocytes and TAP1expression within growing tumors in mice. A. CD4 and CD6 T-cells intumors were detected by FACS analysis using monoclonal antibodies. RM4-5(against CD4) and 53-6.7 (against CD8). a—%=100% X Number of CDs/Numberof total cells (including tumor cells). b. TAP1 expression in vivotumors or cell lines was detected by Western blot using C90 rabbit serumspecific for rate and mice TAP1.

FIG. 39 is bar graph showing the examination of the survival of micebearing CMT tumors. A rTAP1 does not improve recognition of CMT.64 innude mice. 5×10⁵ cells of either CMT1-4 (5 mice) or CMT.neo (4 mice)were injected ip. To nude mice (H-2^(b)). The time of mortidity wasrecorded for each group. B. Immunization with rTAP-transfected tumorsimproves the survival of CMT.64-bearing mice. 1×10⁷ cells of eitherCMT.neo, CMT.1-4 or CMT.1-10 were treated with Mitomycin C (30 mg/ml)for 2 hours and γ-irradiated (10,000 Rads) before injection ip. into 10C57B1/6 mice. One month later the mice were challenged ip. with 5×10⁵CMT.64 cells in PBS. The time of morbidity was recorded.

FIG. 40 is graph showing anti-tumor immune therapy by VV-rTAP1. A. Eachgroup of mice were injected ip. with CMT.neo cells. Two out of threegroups subsequently received twice treatments of either VV-pJS5 orVV-rTAP1 with 106 pfu in PBS containing 2% mouse serum at 24 hours andat 2 weeks after cell injection. Control group received only PBScontaining 2% mouse serum. Statistical analysis shows that VV-rTAP1treatment of tumor-bearing mice has a significant P-value (P<0.05),comparing with both VVVV-pJS5 and mimic treatment. B. The specificity ofsplenocytes from two mice injected with CMT.neo and VV-rTAP1 wasdetermined in a chromium release assay against the ⁵¹Cr-labeled targets.CMT.neo, CMT.1-4, and VV-pJS95 infected CMT.1-4 (10:1 m.o.i infectionfor 3.5 hours).

DETAILED DESCRIPTION OF THE INVENTION I. Therapeutic Methods

The present inventors have surprisingly shown that TAP-1 alone (in theabsence of TAP-2) or TAP-2 alone (in the absence of TAP-1) is sufficientto enhance processing and presentation of VSV peptides to theintracellular site of MHC assembly, permitting stable MHC class Imolecule endogenous peptide complexes to be formed, transported andexpressed at the cell surface. They also demonstrated that TAP-2 alonein the absence of TAP-1, is sufficient to enhance processing andpresentation of the influenza NP366-374 peptide. The inventors have alsoshown that TAP-1 and/or TAP-2 can enhance the immunogenicity of tumorcells.

In summary, the inventors have shown that the TAP molecules act as anadjuvant that can increase the immunogenicity of targets bearing anantigen. Importantly, the inventors have shown that when TAP is includedin a viral vaccine over 10,000 fold less virus can be used to elicit anequivalent immune response that is observed in the absence of TAP. Thishas important implications as it can allow the use of lower doses ofantigen. Using a lower dose of a vaccine increases the safety andreduces the cost per patient.

Accordingly, the present invention provides a method of enhancing animmune response to antigen comprising administering, to a cell or animalin need thereof, an effective amount of an agent that can augment thelevel of a TAP molecule in a target cell bearing the antigen.

The term “enhancing an immune response” means that the method of theinvention evokes and/or enhances any response of the animal's immunesystem, including of either a cell-mediated (i.e. cytotoxic T lymphocytemediated) or humoral (i.e. antibody mediated) response. These immuneresponses can be assessed by number of in vitro or in vivo assays wellknown to those skilled in the art including, but not limited to,cytotoxic T lymphocyte assays, productions of cytokines, regression oftumors, survival of tumor bearing animals, and antibody assays.

The term “TAP molecule” as used herein includes the TAP-1 moleculealone, the TAP-2 molecule alone as well as combinations of both TAP-1and TAP-2. Augmenting either or both of these molecules may be useful inthe method of the invention. One of skill in the art can readilydetermine whether or not one or both need to be augmented in order toincrease the immunogenicity of a particular antigen.

The term “an agent that can augment the level of a TAP molecule” meansany agent that can increase the level or activity of a TAP-1 and/orTAP-2 molecule as compared to the level or activity in same target cellin the absence of the agent. The levels of the TAP-1 and/or TAP-2molecules in the target cell can be readily determined by one of skillin the art using known methods including Western blotting, SDS-PAGE,immunocytochemistry, RT PCR, Northern blotting, and in situhybridization.

The levels of the TAP molecule may be augmented using agents that canincrease TAP expression including interferon-γ and p53. The levels ofthe TAP molecule may also be augmented by administering a nucleic acidmolecule encoding the TAP molecule.

The term “effective amount” as used herein means an amount effective, atdosages and for periods of time necessary to achieve the desired result.The effective amount of a compound of the invention may vary accordingto factors such as the disease state, age, sex, and weight of theanimal. Dosage regima may be adjusted to provide the optimum therapeuticresponse. For example, several divided doses may be administered dailyor the dose may be proportionally reduced as indicated by the exigenciesof the therapeutic situation.

The antigen can be any antigen to which one wishes to generate an immuneresponse including, but not limited to, antigens associated withinfectious diseases (e.g. viral antigens, bacterial antigens, parasiticantigens), self antigens (e.g. antigens implicated in autoimmunediseases) and tumor antigens. The term “tumor antigens” used hereinincludes both tumor associated antigens and tumor specific antigens. Theterm “tumor associated antigens” means an antigen that is expressed onthe surface of a tumor cell in higher amounts than is observed in normalcells or an antigen that is expressed in normal cells during fetaldevelopment. A “tumor specific antigen” is an antigen that is unique totumor cells and is not expressed on normal cells.

Examples of viral antigens include vesicular stomatitis virus (VSV)antigens, influenza antigens. Sendai virus antigens. HIV antigens (e.g.Gag, POL), CMV antigens, Hepatitis B and C antigens, Human PapillomaVirus (HPV) E6 and E7 antigens.

Examples of tumor antigens include carcinoembryonic antigens (CEA),carcinoma associated mutated mucins, for example. MUC-1 gene products,gp100, MART-1/Melan A. gp75 (TRP-1) antigens of MAGE family, forexample. MAGE-1,2,3,4,6, and 12, antigens of BAGE family, antigens ofGAGE family, for example. GAGE-1,2, antigens of RAGE family, forexample, RAGE-1. N-acetylglucosaminyltransferase-V, p15; tumor specificmutated antigens; mutated β-catenin, mutated MUM-1 and mutated cyclindependent kinases-4 (CDK4), mutated oncogene products: p21 ras. BCR-abl,p53 and p185 HER2/neu, mutated epidermal growth factor receptor (EGFR)EBNA gene products of EBV, for example, EBNA-1 gene product E7. E6proteins of human papillomavirus, prostate specific antigens (PSA)prostate specific membrane antigen (PSMA). PCTA-1, idiotypic epitopes orantigens, for example, immunoglobulin idiotypes or T cell receptoridiotypes.

The target cell can be any cell to which one wishes to generate animmune response. When the method of the invention is used in aprophylactic therapy or a vaccine the target all is essentially a normalcell (expressing normal TAP levels) that may not have been otherwiseexposed to the antigen. In such a case, the agent that augments TAP isco-administered with the antigen to which one wishes to generate animmune response. When the method of the invention is used as atherapeutic, the target cell may be previously infected with a pathogen(such as a virus or bacteria) or may be a cell that is malignant orcancerous.

As hereinbefore mentioned, in a preferred embodiment, the method of theinvention involves administering a nucleic acid molecule encoding a TAPmolecule in order to augment the level of TAP expression in the targetcell.

Accordingly, in one embodiment, the present invention provides a methodof enhancing an immune response to an antigen comprising administeringan effective amount of a nucleic acid molecule comprising a sequenceencoding a TAP molecule to an animal or cell in need thereof.

The term “animal” as used herein includes all members of the animalkingdom, including humans. Preferably, the animal to be treated is ahuman.

The nucleic acid molecule encoding the TAP molecule can be administeredto the animal in vivo where the TAP molecule will be expressed in vivo.When administered in vivo, the TAP molecule can be administered by anyroute including, but not limited to, intraperitoneally, intravenously,intratumorally, subcutaneously, orally, mucosally, intradermally orsubmucosally. As an alternative, the TAP molecule can be administered tothe target cells ex vivo where the TAP molecule will be expressed in thecells in vitro and then the target cells expressing TAP can beadministered to the animal.

The nucleic acid molecule comprising a sequence encoding TAP-1 and/orTAP-2 under control of a suitable promoter may be readily synthesizedusing techniques known in the art. A sequence encoding TAP-1 includes asequence encoding a protein having the amino acid sequence as set out inTrowsdale. J. et al., Nature 348:741, 1990 and international ApplicationNo. PCT/US91/06105 published on Mar. 19, 1992. A nucleic acid moleculecomprising a sequence encoding TAP-1 may be isolated and sequenced, forexample, by synthesizing cDNAs from RNA— and using rapid amplificationof cDNA ends (RACE. Frohman, et al., 1986) using oligonucleotidesspecific for TAP-1, and analysing the sequences of the clones obtainedfollowing amplification. Oligonucleotides specific for TAP-1 may beidentified by comparing the nucleic acid sequence of the nucleic acidmolecules of the invention to known sequences of TAP-1. Nucleic acidmolecules used in the method of the invention encoding TAP-1 or TAP-2may also be constructed by chemical synthesis and enzymatic ligationreactions using procedures known in the art. The sequence encoding TAP-1or TAP-2 may also be prepared using recombinant DNA methods.

The method of the invention not only contemplates the use of the knownTAP-1 and TAP-2 sequences but also includes the use of: sequences thathave substantial seqeuence homology to the known TAP sequences,sequences that hybridize to the known TAP sequences as well as allanalogs or modified forms of the known TAP sequences.

The term “sequence that has substantial sequence homology” means thosenucleic acid sequences which have slight or inconsequential sequencevariations from the known TAP sequences i.e., the sequences function insubstantially the same manner and can be used to augment an immuneresponse. The variations may be attributable to local mutations orstructural modifications. Nucleic acid sequences having substantialhomology include nucleic acid sequences having at least 65%, morepreferably at least 85%, and most preferably 90-95% identity with theknown nucleic acid sequences of TAP.

The term “sequence that hybridizes” means a nucleic acid sequence thatcan hybridize to a TAP sequence under stringent hybridizationconditions. Appropriate “stringent hybridization conditions” whichpromote DNA hybridization are known to those skilled in the art, or maybe found in Current Protocols in Molecular Biology, John Wiley & Sons,N.Y. (1989), 6.3.1-6.3.6. For example, the following may be employed:6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by awash of 2.0×SSC at 50° C.; 0.2×SSC at 50° C. to 65° C.; or 2.0×SSC at44° C. to 50° C. The stringency may be selected based on the conditionsused in the wash step. For example, the salt concentration in the washstep can be selected from a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be at high stringencyconditions, at about 65° C.

The term “a nucleic acid sequence which is an analog” means a nucleicacid sequence which has been modified as compared to the known sequenceof a TAP molecule wherein the modification does not alter the utility ofthe sequence as described herein. The modified sequence or analog mayhave improved properties over the known sequence. One example of amodification to prepare an analog is to replace one of the naturallyoccurring bases (i.e. adenine, guanine, cytosine or thymidine) of theknown sequence with a modified base such as such as xanthine,hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyladenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosineand 6-azo thymine, pseudo uracil, 4-thiouracil, 8-halo adenine,8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8-substituted adenines, 8-halo guanines, 8 aminoguanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine andother 8-substituted guanines, other aza and deaza uracils, thymidines,cytosines, adenines, or guanines, 5-trifluoromethyl uracil and5-trifluoro cytosine.

Another example of a modification is to include modified phosphorous oroxygen heteroatoms in the phosphate backbone, short chain alkyl orcycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages in the nucleic acid molecule. Forexample, the nucleic acid sequences may contain phosphorothioates,phosphotriesters, methyl phosphonates, and phosphorodithioates.

A further example of an analog of a nucleic acid molecule of theinvention is a peptide nucleic acid (PNA) wherein the deoxyribose (orribose) phosphate backbone in the DNA (or RNA), is replaced with apolyamide backbone which is similar to that found in peptides (P. E.Nielsen, et al Science 1991, 254, 1497). PNA analogs have been shown tobe resistant to degradation by enzymes and to have extended lives invivo and in vitro. PNAs also bind stronger to a complimentary DNAsequence due to the lack of charge repulsion between the PNA strand andthe DNA strand. Other nucleic acid analogs may contain nucleotidescontaining polymer backbones, cyclic backbones, or acyclic backbones.For example, the nucleotides may have morpholino backbone structures(U.S. Pat. No. 5,034,506). The analogs may also contain groups such asreporter groups, a group for improving the pharmacokinetic orpharmacodynamic properties of nucleic acid sequence.

Some of the methods contemplated herein use nucleic acid moleculescontaining sequences encoding truncated non functional forms of TAP-1 orTAP-2. Truncated non functional forms of TAP-1 and TAP-2 may beidentified by deleting portions of the TAP-1 or TAP-2 gene to producefragments. Such fragments should hybridize to the TAP-1 or TAP-2sequences under stringent hybridization conditions. Stringenthybridization conditions are those which are stringent enough to providespecificity, reduce the number of mismatches and yet are sufficientlyflexible to allow formation of stable hybrids at an acceptable rate.Such conditions are known to those skilled in the art and are described,for example, in Sambrook, et al, (1989, molecular Cloning, A LaboratoryManual, Cold Spring Harbor). The ability of the truncated forms of TAP-1and TAP-2 to transport endogenous peptides may be determined using themethods described herein.

Nucleic acid molecules having a sequence which codes for TAP-1 or TAP-2,including the homologs and modified forms discussed above, may beincorporated in a known manner into an appropriate expression vectorwhich ensures good expression of the protein or part thereof. Possibleexpression vectors include but are not limited to cosmids, plasmids(including both naked DNA plasmids and liposome encapsulated plasmids),or modified viruses, so long as the vector is compatible with the targetcell used.

It is contemplated that the nucleic acid molecules described hereincontain the necessary elements for the transcription and translation ofthe inserted sequence. Suitable transcription and translation elementsmay be derived from a variety of sources, including bacterial, fungal,viral, mammalian, or insect genes. Selection of appropriatetranscription and translation elements is dependent on the target cellchosen as discussed below, and may be readily accomplished by one ofordinary skill in the art. Examples of such elements include: atranscriptional promoter and enhancer or RNA polymerase bindingsequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other genetic elements, such as an origin ofreplication, additional DNA restriction sites, enhancers, and sequencesconferring inducibility of transcription may be incorporated into theexpression vector. It will also be appreciated that the necessarytranscriptional and translation elements may be supplied by the nativeTAP-1 gene, TAP-2 gene and/or their flanking regions.

The nucleic acid molecules may also contain a reporter gene whichfacilitates the selection of transformed or transfected host cells.Examples of reporter genes are genes encoding a protein such asβ-galactosidase, chloramphenicol acetyltransferase, firefly luciferase,or an immunoglobulin or portion thereof such as the Fc portion of animmunoglobulin preferably IgG. In a preferred embodiment, the reportergene is lac Z. Transcription of the reporter gene is monitored bychanges in the concentration of the reporter protein such asβ-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. This makes it possible to visualize and assay for expressionof TAP-1.

Nucleic acid molecules comprising a sequence encoding TAP-1 or TAP-2 canbe introduced into target cells via transformation, transfection,infection, electroporation etc. Methods for transforming transfecting,etc. host cells to express foreign DNA are well known in the art (see,e.g., ltakura et al., U.S. Pat. No. 4,704,362; Hinnen et al., PNAS USA75:19291933, 1978; Murray et al., U.S. Pat. No. 4,801,542; Upshall etal., U.S. Pat. No. 4,935,349; Hagen et al., U.S. Pat. No. 4,784,950;Axel et al., U.S. Pat. No. 4,399,216; Goeddel et al., U.S. Pat. No.4,766,075; and Sambrook et al. Molecular Cloning A Laboratory Manual,2nd edition, Cold Spring Harbor Laboratory Press, 1989, all of which areincorporated herein by reference).

Suitable expression vectors for directing expression in mammalian cellsgenerally include a promoter, as well as other transcriptional andtranslational control sequences. Common promoters include SV40. MMTV,metallothionein-1, adenovirus Ela. CmV, immediate early, immunoglobulinheavy chain promoter and enhancer, and RSV-LTR. Protocols for thetransfection of mammalian cells are well known to those of ordinaryskill in the art.

In a preferred embodiment, the nucleic acid molecule is introduced intothe target cell in a viral vector, preferably vaccinia viral vectors,andenovirus based vectors, lenti virus based vectors and herpes simplexvirus based vectors. The vectors may be live, attenuated, replicationconditional or replication deficient. Most preferably the viral vectorsare attenuated. Suitable promoters for use with vaccinia viruses includeP7.5 (Cochran, M. A. at al, 1985, J. Virol. 54:30), P11 (Bertholet, C.et al, 1985, Proc. Natl. Acad. Sci. USA 82:2096), CAE-1 (Patel, D. D. etal, 1988, Proc. Natl. Acad. Sci. USA 85:9431).

The nucleic acid molecule may be inserted into a non-essential site of avaccinia viral vector. Such non-essential sites are well known and aredescribed, for example, in Perkus et al, 1986. Virology 152:285; Hrubyet al, 1983, Proc. Nacl. Acad. Sci. USA 80: 3411 and; Weir and Moss,1983, J. Virol. 46:530). Recombinant viruses expressing TAP-1 may bereadily identified using techniques known in the art and discussed, forexample, in Moss, B, 1992, (Curr. Topics Microbiol. Immunol. 158:25).

In a preferred embodiment, the nucleic acid molecule comprising asequence encoding a TAP molecule is co-administered with an additionalnucleic acid molecule comprising a sequence encoding an antigenicpeptide, such as a pathogenic peptide or tumor antigen. Examples ofpathogenic peptides include viral peptides such as the ones hereinbeforementioned. Administering the nucleic acid sequence encoding a TAPmolecule with an antigenic peptide will increase the immune response tothe antigen.

The nucleic acid sequence encoding a TAP molecule can also beadministered in conjunction with other stimulatory molecules includinggrowth factors, accessory molecules, anti-angiogenic therapies andchemokines. Examples of growth factors include interferon-γ, IL-1. IL-2,and GM-CSF. Examples of accessory molecules include B7 and ICAM as wellas accessory molecules involved in antigen presentation such as tapasin,calnexin, calreticulin, p58. MHC class I heavy chain, β₂M, LMP2 andother interferon inducible genes. Examples of chemokines includemolecules such as MCP1. These molecules will help stimulate theexpansion of lymphocytes involved in the specific responses that wereinitiated by enhanced MHC class I restriction antigen process in that itoccurs when TAP is expressed above normal levels. Other stimulatorymolecules include genes that are inducible by retinoic acid, tumornecrosis factor, interferon alpha, beta or gamma, tapasin, calnexin,calreticulin, p53, p58, MHC I heavy chain. HSP 70, HSP 90, BIP, GRB94,interferon response proteins 3 and 7.

The additional molecules can either by administered in the form of anucleic acid or as a protein. When administered as a nucleic acid, thestimulatory molecules may be administered as a chimeric nucleic acidconstruct that encodes the TAP molecule, the antigen as well as thestimulatory molecule. In addition, each of the molecules may beadministered in different constructs and administered in differentvectors.

The method of the present invention can be used as an adjunct tosurgery, chemotherapy, radiation therapy, immunotherapy or photodynamictherapy as one of its uses is to treat cancer.

In one embodiment, the target cell is one that normally expresses low ornon-detectable levels of MHC Class I molecules and low or non-detectablelevels of TAP-1 and/or TAP-2 proteins. Such a phenotype is common inmalignant cells.

Accordingly, the present invention provides a method of enhancingexpression of MHC class I molecules bearing endogenous peptides on thesurface of a target cell expressing low or nondetectable levels of MHCclass 1 molecules and possibly expressing low or nondetectable levels ofTAP-1 and TAP-2 transporter proteins comprising: introducing into thetarget cell a nucleic acid molecule comprising a sequence encoding TAP-1or TAP-2 under control of a suitable promoter and; expressing TAP-1 orTAP-2 in the target cell under suitable conditions, thereby enhancingprocessing and presentation of MHC class I molecules bearing endogenouspeptides.

Target cells expressing low or non-detectable levels of MHC Class Imolecules and expressing low or nondetectable levels of TAP-1 and TAP-2transporter proteins may be selected by methods known in the art. Forexample, a target cell may be infected with a recombinant viral vectorsuch as VSV, and tested for lysis by VSV specific cytolytic T cells.FACS analysis may also be used to detect MHC class I molecules on thesurface of a putative target cell. The biosynthesis and intracellulartransport of MHC class I molecules may also be biochemicallycharacterized. For example, endo H which cleaves N-linkedoligosaccharides only when they are in the high mannose formcharacteristic of proteins present in the ER and cis-Golgi complex maybe used to measure intracellular transport. Pulse-chase methodology mayalso be utilized to confirm target cells expressing low levels of MHCclass I molecules. The above methods are illustrated in the Examplesherein. See also Restifo. N. P. et al, supra, 1993.

Examples of cells which express low levels of MHC class I molecules aretumor cells derived from colon, breast, lung mesothelioma and lungcancers of the small cell histology (See Restifo, N. P., supra 1993).

Cells expressing low or nondetectable levels of TAP-1 and TAP-2transporter proteins may be detected by assaying for mRNA encoding theseproteins, for example using Northern Blot analysis as described in theExamples herein. Examples of cells which express low levels of TAP-1 andTAP-2 are tumor cells derived from lung cancers of the small cellhistology.

Target cells may, in addition to expressing low or nondetectable levelsof the transporter proteins TAP-1 and TAP-2, express low ornondetectable levels of one or more of the components of the proteasome,for example LMP7 and LMP-2.

The present inventors have demonstrated that TAP-1 or TAP-2 alone canenhance expression of MHC class I molecule-endogenous peptide complexeson the surface of tumor cells, thereby rendering the tumor cellssusceptible to immune surveillance by CTL.

Accordingly, the present invention provides a method of augmenting theimmune response of a mammal to a tumor cell comprising: introducing anucleic acid molecule comprising a sequence encoding a TAP molecule intothe tumor cell under control of a suitable promoter and; expressing theTAP molecule in the tumor cell under suitable conditions, therebyenhancing the immune response to tumor cell. For the treatment oftumors, the TAP molecule can be administered directly in vivo or can beused to transfect tumor cells ex vivo which are re-infused into thepatient. When the TAP molecule is administered directly in vivo it canbe administered by any route including, but not limited to,intraperitoneally, intravenously, intratumorally, subcutaneously,intradermally, mucosally, submucosally or orally.

In a preferred embodiment, the method further comprises: introducing anadditional nucleic acid molecule into the tumor cell, said additionalnucleic acid molecule comprising a sequence encoding an antigenicpeptide under control of a suitable promoter and; expressing theantigenic peptide in the tumor cell under suitable conditions, therebyenhancing presentation and processing of the antigenic peptidepermitting recognition by the mammal's immune response.

The invention still further relates to a method of preparing tumorspecific T cells which have anti-tumor properties comprising removingtumor cells from a subject; introducing a nucleic acid molecule encodingTAP-1 or TAP-2 under the control of a suitable promoter into the tumorcells; implanting the tumor cells in the subject or a mammal have areconstituted immune system of the subject; and harvesting tumorspecific T cells. It will be appreciated that in an embodiment thenucleic acid molecule may also encode both TAP-1 and TAP-2 or thatseparate nucleic acid molecules encoding TAP-1 and TAP-2 may beintroduced into the tumor cells. The tumor specific T cells may be usedas a therapeutic agent in vivo in the subject. Methods such as thosedescribed in Restifo. N. P. et al J. Exp. Med. 175:1423-1431 may be usedto prepare specific T cells which anti-tumor properties in vivo usingtumor cells transfected with IFN-γ. Adoptive immunotherapy models can beused to confirm the utility of the preparation against establishednon-modified tumor cells in vivo.

The invention also contemplates that nucleic acid molecules encodingTAP-1 and/or TAP-2 may be incorporated into recombinant viral vectorvaccines for use in augmenting the immune response to a pathogen ortumor. Such vaccines can be used to treat infectious agents or to treatcancers. Such vaccines are expected to have particularly usefulapplication for mammals, including humans, which are unable to mount animmune response to certain viral or tumor antigens or where their HLAmakeup does not permit adequate processing and presentation of therelevant antigenic peptide. For example, for use in persons or for tumorcells lacking in components of the antigen presentation system, such asTAP-1, TAP-2 and proteasome components. It will be appreciated that thenucleotide sequences encoding TAP-1 and TAP-2 may be used separately ormay be included together, either under the control of separate promotersor under the control of the same promoter.

Recombinant vaccinia virus vaccines may be constructed using techniquesknown in the art. For example, the pJS5 shuttle vector which containstwo early/late compound promoters may be used to express both TAP andthe relevant antigen in an infected cell simultaneously. The TAP gene orgenes may be cloned behind one promoter and the protein or peptide genecan be cloned behind the second promoter, or in a second vaccine. TAP-1may be cloned behind one promoter and TAP-2 may be cloned behind asecond promoter. The cloned genes may be flanked by the thymidine kinasegene. The pJS5-TAPantigen vector can be transfected into a vacciniainfected cell so the homologous recombination can occur between thethymidine kinase sequence in both vaccinia and the cloned shuttlevector, resulting in either a recombinant vaccinia virus containing TAPand the antigen, or a recombinant vaccinia virus containing both TAP-1and TAP-2 which would allow particular peptides to be transported andpresented.

The invention also contemplates a method for inhibiting rejection by arecipient animal of a transplanted tissue comprising modifying,eliminating, or masking expression of TAP-1. TAP-2 or both TAP-1 andTAP-2 in cells of said tissue to inhibit endogenous antigen processingand presentation on the surface of cells of said tissue which cause aT-lymphocyte mediated response in said animal. Expression of TAP-1,TAP-2 or TAP-1 and TAP-2 may be modified, eliminated or masked usingTAP-1. TAP-2 and/or TAP-1 and TAP-2 antisense. Class I MHC molecules mayalso be eliminated from the cells of the transplant tissue and truncatedforms of TAP-1. TAP-2 and/or TAP-1 and TAP-2 may be used to compete withthe functional transporters resulting in down-regulation of expressionof TAP-1 and/or TAP-2.

II. Compositions

The present invention also includes pharmaceutical compositions orvaccines for carrying out the methods of the invention. Accordingly, thepresent invention provides a pharmaceutical composition for use inenhancing an immune response comprising an effective amount of an agentthat can augment the level of a TAP molecule in admixture with asuitable diluent or carrier. In a preferred embodiment, thepharmaceutical composition comprises an effective amount of a nucleicacid molecule comprising a sequence encoding a TAP molecule in admixturewith a suitable diluent or carrier.

The above described nucleic acid molecules encoding a TAP molecule or avector comprising the nucleic acid molecules may be formulated intopharmaceutical compositions for administration to subjects in abiologically compatible form suitable for administration in vivo. By“biologically compatible form suitable for administration in vivo” ismeant a form of the substance to be administered in which any toxiceffects are outweighed by the therapeutic effects. The substances may beadministered to living organisms including humans, and animals.

The vaccines or pharmaceutical compositions can contain other moleculessuch as the antigen to which one wishes to generate an immune responseand/or stimulatory molecules as hereinbefore described.

The pharmaceutical composition may be administered in a convenientmanner such as by injection (subcutaneous, intravenous, intraperitoneal,intratumoral etc.), oral administration, inhalation, transdermalapplication, or rectal administration. Depending on the route ofadministration, the nucleic acid molecules may be coated in a materialto protect the compound from the action of enzymes, acids and othernatural conditions which may inactivate the compound.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionswhich can be administered to subjects, such that an effective quantityof the active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985) or Handbook ofPharmaceutical Additives (compiled by Michael and Irene Ash, GowerPublishing Limited, Aldershot, England (1995)). On this basis, thecompositions include, albeit not exclusively, solutions of thesubstances in association with one or more pharmaceutically acceptablevehicles or diluents, and may be contained in buffered solutions with asuitable pH and/or be iso-osmotic with physiological fluids. In thisregard, reference can be made to U.S. Pat. No. 5,843,456. As will alsobe appreciated by those skilled, administration of substances describedherein may be by an inactive viral carrier.

The following examples are offered by way of illustration only, and notby way of limitation.

EXAMPLES

The following materials and methods were utilized in the investigationsoutlined in Examples 1 to 8.

Animals and Viruses

C57Bl/6 mice were bred at the University of British Columbia breedingfacility. Mice were 6-12 weeks old and were maintained in accordancewith the guidelines of the Canadian Council on Animal Care. VSV wasgrown on vero cell monolayers. Vaccinia and a human β₂M (hβ₂m) vacciniarecombinant were gifts from Dr. J. Yewdell.

Cell Lines and Antibodies

CMT.64 cells (H-2b), were provided by Dr. L. M. Franks (Franks, L. M. etal 1976, Cancer. Res. 36:1049). RMA and RMA-S cells were maintained inDMEM supplemented with 10% heat activated FCS, 20 Mm Hepes, 2 mMglutamine, and antibiotics. The mABS used were as follows: 142-23.3 antiH-2 K^(b), 28-11-5s anti H-2 D^(b) (α1+α2), 28-14-8s anti H-2 D^(b) (α3)and BBM.1 against human β₂m (Brodsky, F. M. et al, 1979, Eur. J.Immunol. 9:536). A rabbit antiserum against hβ₂M (Bikoff. E. K et al,1991. Nature 354:235), against exon-8 of H-2K^(b) (Williams, D. et al,1989.J. Immunol. 142:2796) and against rat proteasome (Brown. M. G. etal, 1991. Nature 353:357) were also used.

Transfection

Transfection of CMT.64 cells with cDNA from rat TAP-1 in the pHbApr-I-neo expression vector (provided by Dr. G. Butcher) was achieved bylipofection (Lipofectin, Gibco BRL, Gaithersburg, Md.) using 10 μg ofDNA. Selection was in 1 mg/ml G418 (Gibco BRL). Positive clones wereselected and screened by Northern blotting for expression of the ratTAP-1 gene. The results obtained with a representative clone arereported (See FIG. 9). As negative controls, clones obtained from avector DNA transfection were analyzed by Northern Blotting. The resultsobtained with a representative clone are reported (See FIG. 9).

Flow Cytometry Analysis

To determine the cell surface expression of MHC class I moleculesfluorescence-activated cell sorter (FACS®) analysis (Becton Dickinson &Co., Mountain View, Calif.) was used. RMA. RMA-S and CMT.64 cells weretreated with or without recombinant murine gamma interferon (IFN-γ) at150-300 units/mil (Genzyme Cytokine Research Products) for 48 hours. Thecells were collected and incubated overnight in medium without FCS, withVSV-N 52-59 peptide (50 μM) and/or hβ₂m (2.5 μg). Peptides werepurchased from the University of Victoria, Peptide Synthesis Facility(Victoria, BC, Canada). The cells were subsequently removed fromculture, washed, and incubated with 1:50 dilution of 142-23.3 ascites,or 200 μl of cell culture supernatant from 28-11-5s and 28-14-8s cellsfor 45 min on ice. After two washes, the cells were incubated with 100μl of 1:20 dilution of goat anti-rabbit, or goat anti-mouse FITCconjugated secondary antibody for another 45 minutes on ice. The sampleswere then fixed in paraformaldehyde (1.5% in phosphate buffered saline)and analyzed on a FACScan® cell sorter using the FACScan®program (BectonDickinson & Co.). Values reported in Table 1 are in linear termsrepresenting the average of 5,000 cells. The corrected value (minus thevalue without first antibodies) is reported.

Cell Labeling, Pulse-Chase Experiments, Immuoprecipitation, IsoelectricFocusing and SDS-PAGE

Cells were washed in MEM medium without methionine 1 hour beforelabeling and labeled with 150 μCi/Ml of ³⁵S methionine for 1 hour or asindicated. For the pulse-chase experiments, cells were labeled 15minutes and then chased with normal medium containing an excess of coldmethionine. Labeled cells were solubilized with 1 ml of 20 Mm Tris-Hcl(pH 7.6) containing 0.12 M NaCl, 4 Mm MgCl₂ and 1% Nonidet P-40,phenylmethylsulfonylfluoride (PMSF, a protease inhibitor) was added to afinal concentration of 20 μg/ml before use. After 15 min on ice,particulate material was removed by centrifugation. The supernatant wasused for immunoprecipitation of labeled antigens. Labeled solubilizedantigens were first precleared with 2 μl of normal rabbit serum for 45min at 4° C. followed by 50 μl of protein A-Sepharose (1:1 insolubilization buffer) for another 45 minutes at 4° C. ProteinA-Sepharose was removed by a quick centrifugation. The preclearedsupernatant was reacted with the appropriate antibody or immune serumfor 1 hour at 4° C. 35 μl of protein A-Sepharose was added andincubation continued for a further 30 minutes. After centrifugation thebeads were washed twice with 0.2% NP-40 in 10 mM Tris-HCl pH 7.5, 0.15 MNaCl and 2 mM EDTA, once with 0.2% NP40 in 10 Mm Tris-HCl, pH 7.5, 0.5 MNaCl, 2 mM EDTA and finally with 10 mM Tris-HCl pH 7.5. One-dimensionalisoelectric focusing was performed as previously described in Celis. J.E. et al (1990. Electrophoresis 11:989). SDS—PAGE was carried out asdescribed in Kvist, S. et al, (1982. Cell 29:61).

CTL Response Against VSV-Infected, IFN-γ Induced Cells

RMA, RMA-S and CMT.64 cells were treated with or without IFN-γ at 200units per ml for 48 hours. They were subsequently washed 3× with PBS andtreated with VSV at a multiplicity of infection (MOI) of 5 min in 0.5 mlof medium for one hour. The cultures were then incubated in a total of 3ml of growth medium for an additional 4-8 hours (as indicated), to allowinfection to proceed. Single cell suspensions were treated with 100 μCi⁵¹Cr per 10⁶ cells for 2 hours in RPMI 1640 supplemented withL-glutamine and penicillin/streptomycin in the absence of fetal bovineserum (FBS) and sodium bicarbonate. Alternatively. CMT.64 cells wereinfected with Vaccinia (V), and/or Vaccinia-β2m (Vb2) at an MOI of 5 for5 hours followed by superinfection with VSV (MOI,5) for an additional 4hours. The cells were washed 3× and subseauently incubated at 10⁴ cellsper well in 96-well plates with the effector population at ratios of 100to 12.5. Mock infected cells were used as negative controls. Theeffector CTL population was generated by immunizing C57B1/6 mice withVSV at 5×10⁶-1×10⁷ TCID₅₀ in the foot pads and ears. On day 5 postimmunization the draining lymph nodes (retropharyngeal and popliteal)were harvested and cultures initiated at 4×10⁶ cells per ml in a totalvolume of 5 ml in 6-well plates. The culture medium consisted ofRPMI-1640 supplemented with 5×10⁻⁵ M 2-mercaptoethanol (ME), 10% heatinactivated FBS, sodium pyruvate, penicillin, streptomycin, L-glutamine.HEPES, sodium bicarbonate, and 50% NCTC-109. Cultures were incubated forthree days at 37° C. and 5% CO₂ in the absence of exogenous stimulation.The ⁵¹Cr release was measured by a compugamma counter (model 1282 CS;LKB Instruments, Gaithersburg, Md.) and the specific ⁵¹Cr releasecalculated as [(experimental−media control)/(total− mediacontrol)]×100%. The spontaneous release never exceeded 17% of themaximum release.

RNA Extraction and Northern Analysis

Total cellular RNA was prepared from cell lines using guanidiniumisothiocyanate (GITC). Briefly, the cells were lysed in 4 M GITC thencentrifuged (130,000 g for 16 hours at 23° C.) through a cushion ofcesium chloride. After ethanol precipitation, the purified RNA wasresuspended in DEPC-treated H₂O. 10 μg of each sample was loaded andseparated on a 1% agarose gel containing 2.2 M formaldehyde. The gel wasblotted onto Hybond N (Amersham Corp., Arlington Heights, Ill.) and U/Vfixed prior to hybridization. The ³²P-labelled probes used forhybridization were as follows MTP1 and MTP2 (TAP-1 and -2 respectively,kindly provided by Dr. Geoff Butcher), prepared by random priming, andan oligonucleotide specific for β-actin labelled by terminaltransferase. Hybridization was carried out at 42° C. in buffercontaining 0.4 M Na₂HPO₄, 50% formamide and 7% SDS. Several washes wereperformed at 42° C. under conditions of increasing stringency and thefilter exposed to X-OMAT AR film (Kodak) overnight.

Example I Comparison of the Phenotypes of CMT.64 and RMA-S Cells

The small lung carcinoma cell line. CMT.64, was shown to express andassemble MHC class I molecules on the cell surface after IFN-γ treatment(Klar D. and Hammerling, 1989, EMBO J. 8:475; Sibille, C. et al, 1992,Eur. J. Immunol. 22:433; and Jefferies W. A. et al. 1993, J. Immunol.151:2974). In order to understand the molecular deficiency in antigenprocessing of CMT.64 cells, the contrasting phenotypes of CMT.64 cellsversus RMA-S cells were analyzed. CTL recognition of VSV infected RMA,RMA-S, CMT.64 IFN-γ induced or uninduced cells was investigatedgenerally following the methods outlined in the methodology sectionherein. More particularly, target cells were treated with or withoutIFN-γ for 48 hours prior to infection with VSV and the results are shownin FIG. 1. Panel A in FIG. 1 illustrates a representative experimentusing RMA and RMA-S cells as targets, whereas panel B is the equivalentexperiment with CMT.64 cells (abbreviated: C). All cells were infectedwith VSV at an MOI of 10 for 4 hours. IFN-γ treatment is denoted in FIG.1 with a + sign following the cell line designation. Spontaneous releasedid not exceed 15%.

VSV infected RMA-S cells are recognized as efficiently as the wild-typeRMA cells with or without IFN-γ treatment (FIG. 1A), in comparison toVSV infected CMT.64 cells which are not recognized by specific CTLunless induced by IFN-γ (FIG. 1B). It should be noted that RMA-S, RMAand CMT.64 cells are equally permissive to infection with VSV asindicated by the number of infective particles produced followinginfection measured by a TCID₅₀ assay (data not shown). Therefore,uninduced CMT.64 cells have a different or additional deficiency to thefunctionally defective peptide transporter TAP-2 present in R×A-S cells.

Example 2 The Effect of Exogenous and Endogenous Peptide on thePhenotypes of CMT.64 and RMA-S Cells

Previous experiments have demonstrated that treatment of mutant cellswith exogenous peptides and/or human β₂m can stabilize “empty” class Imolecules at the cell surface (Townsend. A. et al. 1989. Nature 340:443;Vitiello, A., et al., 1990. Science 250:1423; and Ljunggren, H.-G., etal. 1990. Nature 346:476). RMA, RMA-S and CMT.64 cells uninduced orinduced with IFN-γ were treated overnight with exogenous peptides VSV-N52-59 at 50 μM in the presence or absence of human β₂m. VSV-N 52-59peptides and β₂m synergistically increase the expression of the K^(b)conformational specific epitope recognized by 142.23.3 mAb (Table 1) onRMA and RMA-S cells. VSV-N 52-59 peptides specifically affect thestability and the conformation of the K^(b) molecules and have no effecton D^(b) molecules. Human β₂m binds to K^(b) and D^(b) molecules, whichis detected by BBM.1 (anti-human β₂m mAb), and appears to stabilizeheavy chains before they can disassemble at the cell surface. Astabilizing effect was not seen after CMT.64 treatment with peptides orβ₂m alone. Additional treatment of CMT.64 cells with IFN-γ was requiredfor high expression of K^(b) and D^(b) conformation specific epitopes onthe cell surface (Table 1). Therefore, CMT.64 cells express much loweramounts of ‘empty’ class I molecules at the cell surface than RMA-Scells.

Earlier work has shown that the presence of β₂m and peptides within thelumen of the ER is necessary for efficient assembly and cell surfaceexpression of MHC class I molecules (Rock, K., et al., 1991. Cell65:611). FIG. 2 shows the amount of β₂m synthesized in RMA, RMA-S andCMT.64 IFN-γ induced or uninduced cells. Cells were labeled for 2 hourswith ³⁵S-methionine lysed, immunoprecipitated with a rabbit anti-hβ₂° m.serum, and analyzed by SDS-PAGE. Radioactive proteins were detectedafter 6 hours exposure to a JAR film. CMT.64 cells treated (+) or not(−) with IFN-γ. RMA and RMA-S cells were used (FIG. 2). The migration ofthe molecular weight marker is indicated on the left of FIG. 2.

As illustrated in FIG. 2, CMT. 64 cells express a low amount ofendogenous β₂m (FIG. 2, lane 1). IFN-γ induced CMT.64 cells express amuch higher amount of β₂m, which is comparable to the level expressed inRMA and RMA-S cells (FIG. 2, lanes 2-4).

To investigate the effect of β₂m in CMT.64 cells, a recombinant vacciniavirus was used to increase the amount of endogenous β₂m. The effect ofβ₂m on the CTL response against CMT.64 cells is shown in FIG. 3.Infected CMT.64 cells were superinfected with Vaccinia and Vaccinia-β₂mrecombinant (V-Vb2), Vaccinia and VSV (V-VSV), or Vaccinia-β₂m and VSV(Vb2-VSV) in FBS free media (MOI 3) for up to an additional 12 hours. Inthe inset in FIG. 3, the level of (β₂m synthesized is shown afterimmunoprecipitation with the anti-hβ₂m rabbit serum. CMT.64 cellstreated with peptide VSV-N52-59 at 500 μM for 2 hours (CMT+p) was usedas the positive control, whereas mock treated CMT.64 cells (CMT+-) wereused as the negative control. Radioactivity released is the average ofquadruplicate wells. Spontaneous release did not exceed 16%.

As illustrated in FIG. 3, elevating the amount of β₂m synthesized usinga recombinant vaccinia virus did not restore CTL recognition of VSVinfected CMT.64 cells. Therefore, increasing expression of β₂m does notinduce presentation of VSV-N peptides in the context of K^(b) molecules.The CMT.64 antigen processing phenotype is not caused by the low amountof endogenous β₂m.

Example 3 Intracellular Transport of MHC Class I Molecules D^(b) andK^(b)

The transport of K^(b) and D^(b) molecules was examined after apulse-chase labelling of CMT.64, RMA and RMA-S cells and SDS-PAGEanalysis of the immunoprecipitated material (for K^(b), 142.23.3 mAb wasused and for D^(b), 28-14-8s an α3 specific mAb was used). Intracellulartransport of MHC class I molecules, D¹⁰ and K^(b) is shown in FIG. 4.Cells were labelled with ³⁵S-methionine and chased in an excess of coldmethionine for the times indicated in hours at the top of FIG. 4.Solubilized antigens were immunoprecipitated with 142.23.3 mAbs forK^(b) or 28-14-8s mAbs for D^(b). Treatments are indicated on top andthe migration of the molecular weight markers is indicated on the leftof FIG. 15. Communoprecipitation of the heavy chains (46 kD) and β₂M (12kD) can be seen for all cells except for CMT.64 uninduced cells.Radioactive proteins were detected after 8 days exposure for RMA-S cellsand 4 days for RMA and CMT.64 cells to a XAR film.

Despite a similar amount of K^(b) molecules synthesized in RMA and RMA-Scells (FIG. 4, 0 hour chase time), only low amounts of K^(b) areprocessed to a higher mol. weight form indicative of the level oftransport which accounts for the surface expression of K^(b) in RMA-Scells. The processed form is resistant to endoglycosidase H digestion(data not shown) indicating transport out of the ER. The observationthat much more β₂M was immunoprecipitated with K^(b) molecules than withD^(b) molecules in RMA-S cells (FIG. 4) may indicate that the mAb142.23.3 only recognizes the assembled form, heavy and light chains ofK^(b) molecules, whereas the mAb 28-14-8s recognizes the α3 region ofD^(b) molecules. The presence of a functional TAP-1 protein in RMA-Scells (Yang. Y. et al., 1992, J. Biol. Chem. 267:11669) may besufficient to enable some peptides to cross the ER membrane and bind asmall number of K^(b) molecules allowing them to go to the cell surface.Also, peptides with lower affinity for K^(b) molecules may bind and aidthe molecules to assemble and go to the cell surface where theydissociate. Much fewer mature processed D^(b) molecules were detected inRMA-S cells after 4 hours chase (FIG. 4). This may indicate a loweraffinity of D^(b) for NM and/or fewer peptides available for D^(b)binding. In RMA cells, K^(b) molecules were processed within 1 hour. Incomparison, D^(b) molecules were processed more slowly (2 hours) (FIG.4). These results are in agreement with the relative transport rate ofK^(b) and D^(b) in RMA-S cells. IFN-γ treatment augments the synthesisof heavy chains causing more K^(b) and D^(b) molecules to be transportedto the cell surface of RMA and RMA-S cells. The rate of transport ofK^(b) and D^(b) molecules was not affected by IFN-γ treatment in RMA andRMA-S cells. In contrast, no K^(b) molecules were detected in CMT.64cells using the 142.23.3 mAb

Example 4 Intracellular Transport of Free and Assembled Forms of K^(b),Molecules in Uninduced and IFN-γ Induced CMT.64 Cells

As discussed above, the 142.23.3 mAb may not recognize the unassembledand peptide free heavy chains of K^(b) molecules (FIG. 4). In order toaddress this issue, a rabbit anti-exon 8 serum directed against aconformation independent epitope recognizing a peptide in thecytoplasmic tail of H-2K^(b) molecules (Williams, D. et al, 1989, J.Immunol. 142:2796) was used to detect and follow the processing of K′molecules in uninduced or IFN-γ induced CMT.64 cells.

Cells were labelled with ³⁵S-methionine and chased in an excess of coldmethionine for the times indicated in hours at the top of FIG. 5.Solubilized antigens were immunoprecipitated with a rabbit anti-exon 8of H-2K^(b) serum recognizing the cytoplasmic tail of free and assembledK^(b) heavy chains as described herein. Treatments are indicated on topand the migration of the molecular weight markers is indicated on theleft of FIG. 5. Radioactive proteins were detected after 4 days exposureto a RPN-30 film (Amersham, Corp.).

In uninduced CMT-64 cells, K^(b) molecules were detectable early aftersynthesis (FIG. 5, 0 h, 0.5 h and 1 h chase time), but were unstable andmostly degraded after 8 h chase with very few molecules processed to ahigher mol. weight (FIG. 5, 8 h chase time). In induced cells. K^(b)molecules were synthesized in higher amounts and a greater proportion ofthe molecules were processed to a higher mol. weight (FIG. 5). Thedecrease in the amount of material immunoprecipitated by this anti-serumduring the chase could not be explained. A loss or degradation of theepitope recognized by the anti-serum during transport is possible.Furthermore, D^(b) molecules are also synthesized and are then degradedor denatured (FIG. 4). In uninduced CMT.64 cells, no processed D^(b)molecules were detected even after 4 hours chase. Only treatment withIFN-γ results in higher expression, increased transport and increasedtransport rate of K^(b) and D^(b) molecules in CMT-64 cells. Thus,components necessary for the assembly and transport of K^(b) heavychains and β₂m were induced by IFN-γ in CMT.64 cells, while similarinduction did not significantly alter the transport of D^(b) and K^(b)in RMA or RMA-S cells. This indicates that CMT.64 cells are likelydeficient in components necessary for MHC class I assembly which differfrom the TAP-2 defect in RMA-S cells.

Example 5 VSV-N 52-59 Peptide Response in CTL Recognition

In order to assay the function of the MHC class I molecules, the CTLrecognition of CMT-64 cells IFN-γ induced or uninduced and RMA-S cellstreated with exogenous peptides was examined. CMT.64 cells (CMT),CMT.64+IFN-γ (CMT+IFN-γ) and RMA-S cells (RMA-S) were treated withpeptide N52-59 at the concentrations indicated in FIG. 6. Theradioactivity released by specific CTL recognition and lysis wasmeasured and represented as indicated in materials and methods describedabove. Radioactivity released, shown in FIG. 6, is the average ofquadruplicate wells. Spontaneous release did not exceed 13%.

In a dose dependent manner. RMA-S and CMT.64 IFN-γ treated cells were10,000 times more sensitive than CMT.64 cells to killing by specific CTLafter 2 hours treatment with exogenous peptides (FIG. 6). These resultsprovide evidence for the low expression of peptide receptive MHC class Imolecules on the surface of uninduced CMT-64 cells. In the dose-responseon RMA-S cells, a maximum of 15,000 peptide molecules per cell wereneeded to achieve 50% killing by specific CTL, whereas a lower thresholdof 150 molecules per cell resulted in the release of 5-10% of ⁵¹Cr.These data may be explained by a high amount of receptive molecules orhigh affinity of the 5 MHC class I molecules for the peptide on thesurface of RMA-S and IFN-γ induced CMT.64 cells. Under the conditions ofthe present assay, where there is no exogenous β₂m, the exogenouslyadded peptides likely stabilize the empty K^(b) molecules which arriveat the cell surface of RMA-S before they dissociate from β₂m (Rock. K.et al, 1991. Cell 65:611 and Jefferies W. et al, supra, 1993). The lowamount of empty Kb transported in uninduced CMT.64 cells would explainthe difference in sensitization to exogenous peptides.

Example 6 Expression of TAP-1 and TAP-2 Genes in CMT. 64 and RMA-S cells

The results shown in FIGS. 2 to 6 indicate that despite its lowerexpression, β₂m alone is not responsible for the lack of antigensynthesized in these cells but very few are transported to the cellsurface where they bind exogenously added peptides. Besides heavy andlight chains, peptides are necessary for the efficient assembly of MHCclass 1 molecules in the ER (Spies, T. et al, 1990, Nature 348:744;Monaco, J. J. et al, 1990. Science 250:1723; Spies, T. and DeMars. R,1991, Nature 351:323 and; Townsend. A. et al, 1989, Nature 340:443). Thepossibility that the absence of components responsible for thegeneration and transport of these peptides within the ER may beresponsible for the CMT.64 phenotype was investigated.

A putative peptide transporter, presumed to be composed of a heterodimerof two half-ABC type transporter 35 called TAP-1 and TAP-2, has beenimplicated in translocating peptides into the ER for MHC class Iassembly (Kelly. A. et al, 1992, Nature 355:641 and; Powis. S. H. et al.1992. Proc. Natl. Acad. Sci. USA 89:1463). To characterize thedifference of phenotypes between RMA-8 and CMT.64 cells, the expressionof TAP-1 and TAP-2 genes in these cell lines was examined by Northernblot analysis of total cellular RNA from RMA, RMA-S, CMT.64 IFN-γinduced and uninduced cells (FIG. 7). 10 μg of cytoplasmic RNA fromCMT.64, RMA and RMA-s cells IFN-γ induced (+) or uninduced (−) wereanalyzed and the results are shown in FIG. 7. TAP-1, TAP-2 and β-actinprobes were hybridized with the membrane. The radioactivity bound tospecific RNA sequences was detected after overnight exposure of themembrane to a XAR film.

In FIG. 7, Northern Blot analysis shows that uninduced CMT-64 cells didnot express a detectable amount of TAP 1 and TAP-2 mRNA and that theamount of these mRNAs was highly increased after IFN-γ treatment ofthese cells. in addition. FIG. 7 shows that no major difference existsbetween TAP-1 and TAP-2 gene expression in RMA-S and RMA cells, and thatIFN-γ treatment only marginally affected TAP-1 and -2 expression inthese cells. The amount of actin mRNA gives an indication of the nearequal amount of mRNA loaded on the gel for Northern blotting. The IFN-γinducibility of TAP-1 and -2 has been previously demonstrated in mousetissues (Gaskin. H. R. et al, 1992. Science.256:1826), however this hasnot been examined in RMA, RMA-S or CMT.64 cells before this study. Theresults reported here show that the TAP-1 and TAP-2 genes are IFN-γinducible in CMT.64 cells and to a lesser degree in RMA and RMA-S cells.The absence of TAP-1 and TAP-2 mRNA expression in CMT-64 cells likelycauses a lack of antigenic peptides in the ER for binding to andassembly of MHC class I molecules. This results in the nonrecognition ofVSV-infected CMT.64 cells. In contrast, RMA-S cells express a functionalTAP-1 molecule that may aid peptides to cross the ER membrane. Thiswould explain the assembly and transport of MHC class I in RMA-S cellsand their CTL recognition after VSV infection. The lack of TAP-1 andTAP-2 in uninduced CMT.64 cells may be one of the factors responsiblefor the phenotype of CMT.64 cells characterized by the formation ofunstable and inefficiently transported MHC class I complexes.

Example 7 Proteasome Components from RMA, RMA-S and CMT.64 IFN-γ Inducedor Uninduced Cells

Before concluding that TAP deficiencies are the likely or only defectsin CMT.64 cells, the presence of proteasome components in these cellswas examined. Viral peptides are thought to be generated in thecytoplasm by the proteasome (Ortiz-Navarette. V. at al., Nature 353:662,1991; Brown, M. G. et al., Nature 353:355, 1991; Glynne, R. et al.,Nature 353:357, 1991; Martinez, C. K. and, J. J. Monaco, Nature 353:664,1991; Kelly, A. et al., Nature 353:667, 1991; Yang, Y. et al., Proc.Natl. Acad. Sci. USA 89:4928, 1992; and Goldberg, A. L. and K. L. Rock,Nature 357:375, 1992) before crossing the ER membrane. The proteasomecomponents are likely key players in antigen processing which could beabsent in these cells. A rabbit anti-rat proteasome serum was used whichrecognizes the mouse proteasome. After immunoprecipitation of theproteasomes, the different component low molecular mass polypeptides(LMP) produced in these mouse cells can be analysed by two dimensionalgel electrophoresis.

Two dimensional gel analysis of proteasome components from RMA, RMA-Sand CMT.64 IFN-γ induced or uninduced cells are shown in FIG. 8. Cellswere labeled for 2 hours with ³⁵S methionine. Solubilized antigens wereimmunoprecipitated with a rabbit anti-rat proteasome serum and analyzedafter isoelectric focusing in a first dimension and 10-15% SDS-PAGE in asecond dimension. The radioactive proteins were detected after 10 daysexposure to a XAR film. Treatment of the cells is indicated on the topof FIG. 8. The acidic side of the gel is on the right and the basic sideis on the left of the gel. The migration of the molecular weight markersis indicated on the left of the gel. The missing proteins are indicatedby an arrow and are numbered (FIG. 8). Proteins numbered 1 and 7correspond to LMP-7 and LMP-2, respectively.

Two dimensional gel analysis of immunoprecipitations revealed that themajor components of the proteasome are not affected by IFN-γ treatmentof CMT.64 cells but that seven components, including LMP-2 and -7, weremissing in uninduced CMT.64 cells. According to the results of others(Fruh. K. et al, 1992, J. Biol. Chem. 267:22131), the proteins numbered1 and 7 in FIG. 8 correspond to LMP-7 and LMP-2, respectively. LMP-2.LMP7 and five other components of the proteasome were upregulatedslightly by IFN-γ in RMA and RMA-S cells and induced from a state of anundetectable expression to a higher detectable level of expression inIFN-γ treated CMT.64 cells (FIG. 8). LMP-7 (FIG. 8, 1) is particularlyhighly induced in CMT.64 cells treated with IFN-γ. These resultscontrast the results of others which suggested that CMT.64 express a lowlevel of all proteasome components (Ortiz-Navarette, V. et al, 1991.Nature 353:662) and these new results indicate that these inducedproteasome components affect the activity of the proteasome and allowthe generation of the VSV-N peptides in induced CMT.64 cells. Recentdata (Arnold, D. et al., 1992. Nature 360:171, and Momburg. F. et al.,1992. Nature 360:174) suggest that LMP-2 and -7 may not be necessary forinfluenza virus antigen presentation in mutant cells transfected withthe TAP-1 and -2 genes.

The above results show that IFN-γ treatment in addition to inducingtranscription of TAP-1 and TAP-2 genes also upregulates the synthesis ofseven components of the proteasomes, including LMP-2 and -7. Othersdescribe that components in addition to LMP-2 and LMP-7 are upregulatedin Hela cell proteasomes by IFN-γ treatment (Yang. Y. et al., Proc.Natl. Acad. Sci. USA 89:4928, 1992; and Fruh, K. et al., J. Biol. Chem.267:22131, 1992). However, as these cells are functionally wild-type,the functional ramification of this regulation has not been addressed.Furthermore, as LMP-2 and LMP-7 are first synthesized as precursorproteins which are cleaved into smalled products (Fruh. K. et al., J.Biol. Chem. 267:22131, 1992), it is possible that some of the fiveadditional proteins missing from uninduced CMT.64 cells may be precursorproteins of LMP-2 and LMP-7.

Example 8

Recognition of VSV infected TAP-1 Positive CMT.64 Cells

Consideration of the accumulated data regarding antigen processing inRMA-S and CMT.64 cells leads to the contention that a functional TAP-1protein homodimer alone may facilitate the transport of the VSV-N 52-59peptide from the cytosol to the ER lumen where binding to the heavychains takes place. An alternative explanation is that this peptide doesnot require a transporter for translocation across the ER membrane butis not generated in the CMT.64 cells. In order to more clearly definethe defect affecting the recognition of VSV infected CMT.64 cells byspecific CTL, the rat TAP-1 gene was introduced in CMT.64 cells.

CMT 64 (CMT), CMT 64 transfected with TAP-1 (CMT TAP 1), and CMT.64cells transfected with the vector only (CMT Vec) were infected with orwithout VSV for 8 hours at MOI of 5, or treated with N52-59 peptide for2 hours at 500 pM (50% dose response). Spontaneous release did notexceed 12%. FIG. 9 shows that VSV infected TAP-1 positive CMT.64 cellswere recognized by specific CTL.

This result explains the RMA-S phenotype and its apparent “leakiness”regarding VSV presentation (Esquivel. F., et al., J. Exp. Med. 175:163,1992; Hosken, N. A. and M. J. Bevan, J. Exp. Med. 175:719, 1992). TAP-1alone appeared to be sufficient for VSV presentation in RMA-S cells andin transfected CMT.64 cells and may form a homodimer capable oftranslocation of specific peptides into the lumen of the ER. In additionto transporters, the difference in the RMA-S and CMT.64 phenotype may beexplained at one level by the higher amount of viral peptides generatedin RMA-S cells. Interestingly it appears that a total repression of theexpression of both LMPs and TAPs localized in the same region of classII may be sufficient for avoidina any expression of class I the cellsurf ace. This may be very important for some cancer cells (Brodsky. F.M. et al., Eur. J. Immunol. 9:536, 1979; Restifo, N. P. et al., J. Exp.Med. 177:265, 1993; and Bikoff. E. K. et al., Eur. J. Immunol. 21:1997,1991) by providing a method by which tumour cells avoidimmunosurveillance.

Example 9 Tap Gene Expression Profiles of CMT.64 and CMT64/R1-4

In order to investigate the ability of TAP-1 to function independentlyin peptide transport, the rat TAP-1 cDNA was introduced into the murinesmall lung carcinoma cell line CMT.64 which does not express endogenousTAP-1 or TAP-2 mRNA (FIG. 10). The endogenous TAP genes, as well asthose coding for the putative proteasome components LMP2 and 7, areexpressed only after IFN-γ treatment (FIG. 10). Positive transfectantswere selected using the neophosphotransferase selection system andconstitutive expression of the TAP-1 gene was confirmed by northernblotting.

In particular, transfection of CMT. 64 cells with rat cDNA TAP-1 (in thepHb APr-1-neo expression vector as described in Powis, S. J. et al.Nature 354, 528-531 (1991)) was achieved by lipofection (Lipofectin,BRL) using 10 μg of DNA. Selection was in 1 mg/ml G418 (Gibco). TotalRNA was isolated using guanidine isothiocyanate and electrophoresed on a1% agarose gel containing 2.2M formaldehyde (10 μg/lane). Blotting andhybridisation with [³²P]-labelled cDNAs (TAP-1 and 2) or oligonucleotide(actin) were carried out as described herein.

Both TAP-1 and 2 mRNA transcripts were absent in uninduced CMT-64 cellsbut were detected in CMT.64 cells cultured in the presence of 200units/ml of mouse recombinant IFN-γ for 48 hours (FIG. 10), CMT64/rl-4expressed high levels of vector-derived TAP-1 mRNA but remained negativefor TAP-2. Actin mRNA was used to demonstrate that equal amounts of RNAhad been loaded.

Three high expressing clones were selected for subsequent experiments(FIG. 10). Three clones transfected with vector alone were also selectedand used as controls in the subsequent experiments.

Example 10 TAP-1 Expression is Sufficient to Increase Levels of K^(b)and at the Cell Surface of CMT.64 Cells

CMT.64 cells also express virtually no surface MHC class 1 molecules,despite synthesis of both D^(b) and K^(b) heavy chains (Jefferies, W. A.et al., supra, 1993). To determine the influence of TAP-1 on surfaceclass I expression, flow cytometry was carried out using antibodiesagainst D^(b) and K^(b). In particular, the cells were incubated with orwithout primary antibody for 1 hour. All incubations were carried out at4° C. After washing, the cells were incubated with fluoresceinisothiocyanate-conjugated goat anti-mouse Ig antibody (20 mg/ml) for anadditional hour. Following two rounds of washing, the cells were fixedin 1.5% paraformaldehyde. The fluorescent profiles were obtained byanalysing 5,000 cells in a semi-logarithmic plot using a FACScan®programme.

Flow cytometric analysis demonstrated that TAP-1 expression wassufficient to increase levels of K^(b) and D^(b) at the cell surface ofCMT.64 cells as shown in FIG. 11. The antibodies 28.14.8s and Y-3recognise D^(b) and K^(b) respectively. In all panels the resultsobtained with the indicated primary antibody are shown by a solid line.The dotted line represents the values obtained in the absence of aprimary antibody. The results shown are representative of threeindependent experiments.

For both antibodies tested there was a detectable increase in surfaceexpression in the CMT-TAP-1 transfectants compared to CMT.64 and thevector controls, suggesting TAP-1 alone had delivered peptides to thesite of MHC assembly, allowing stable complexes to be formed,transported and expressed at the cell surface. The amount of transferrinreceptor expressed at the cell surface was unchanged by the transfectionof TAP-1, indicating that this was not a general effect on plasmamembrane proteins (data not shown). In contrast to other systems whereit has not been possible to discount the involvement alternativemechanism of peptide transport (Hosken, N. A. & Bevan, M. J. J. Exp.Med. 175:719-729 1992; Esquivel, 5 F., Yewdell, J. & Bennink, J. J. Exp.Med. 175:163-168, 1992; Zweerink, H. J., et al. J. Immunol.150:1763-1771, 1993), these results clearly demonstrate the ability ofTAP-1 to increase surface class I expression in the absence of TAP-2 inCMT.64 cells.

Example 11 Pulse-Chase Analysis of K^(b) and D^(b) Molecules from CMT.64and TAP-1 Transfected CMT.64 Cells

Intracellular transport of class I heavy chain to the cell surface isaccompanied by processing to a higher molecular weight form bymodification of the N-linked glycans during successive exposure toGolgi-specific enzymes. Pulse-chase experiments were therefore performedto determine if such processing was achieved in the CMT-TAP-1transfectants, as predicted by the increase in surface expression ofK^(b) and D^(b).

Pulse-chase and immunoprecipitation of K^(b) and D^(b) were performedusing a 15 minute pulse with ³⁵S-methionine (Amersham) andimmunoprecipitated 28.14.8S (anti-D^(b)) and Y-3 (anti-K^(b)) monoclonalantibodies, following the methods described above. Samples were analysedby SDS-PAGE on 10-15% gels and treated with an amplifying solution. Theautoradiogram was developed after 10 days.

Transport of K^(b) and D^(b) molecules to the cell surface occurred inthe TAP-1 transfected cells, as indicated by the increase in molecularweight of the heavy chain during oligosaccharide side chain processing(FIG. 12). In untransfected CMT.64 cells no processing was observed(FIG. 12), indicating retention within the endoplasmic reticulum or cisgolgi. This was confirmed by sensitivity to endoglycosidase H (data notshown).

In summary, comparison between CMT.64 and CMT64/rl-4 revealed that thepresence of TAP-1 was sufficient to allow the processing of D^(b) andK^(b) to occur (FIG. 12). In addition, it was determined byendoglycosidase H treatment that the higher molecular weight processedforms of Kb and Db were resistant to digestion (data not shown). Theseresults further confirm the importance of TAP-1 for the transport andsurface expression of MHC class I molecules in these cells.

Example 12 TAP-1 Transfected CMT.64 Cells Efficiently Present Antigen toVSV Specific CTL

In previous studies it was established that CMT.64 cells were unable topresent VSV peptides to cytolytic T lymphocytes (CTL) unless pretreatedwith IFN-γ or incubated directly with a synthetic peptide derived fromthe VSV N protein (Jefferies, et al. J. Immunol., 151:2974-2985, 1993).To examine the ability of TAP-1 expression to complement functionalantigen processing and presentation, chromium release assays werecarried out with VSV-specific CTL using CMT.64 and CMT-TAP-1 and CMTvector transfectants as targets.

In particular, CMT.64 cells (CMT) and CMT-TAP-1 transfectants wereinfected with VSV (MOI:2) for 8-10 hours, or treated with Influenzastrain A/PR/8/34 for 48 hours (at 300 HA units for RMA, and 500 HA unitsfor CMT.64 and their derivatives). Effector CTL populations weregenerated by infecting C57bl/6 mice with VSV in the foot pads and earsor 700 HA units of Influenza i.p. VSV CTL were derived from draininglymph nodes as collected on day 5 post immunization and single cellsuspensions were cultured at 4×10⁶ cells per ml for 3 days in theabsence of any restimulation. Influenza CTL were derived fromsplenocytes, 4-5 weeks post-immunization, cultured in the presence ofinfluenza infected stimulators for 6 days. The culture medium consistedof a 1:1 ratio of RPMI-1640 and NCTC-109 supplemented with 10% FBS,L-Glutamine, Pen/Strep, and 2 ME. Targets and effectors were mixed andincubated for 4 hours. Mock infected cells (+-) were used as negativecontrols. The results are expressed as % specific release, as detailedin Jefferies, W. A., Kolaitis, G. & Gabathuler, R. J. Immunol. 151,2974-2985 (1993).

The results illustrated in FIG. 13 show that TAP-1 transfected CMT.64cells (clones CMT-r1.1, r1-4 and r1-10) efficiently present antigen toVSV specific CTL. Introduction of TAP-1 into CMT.64 cells restoresantigen presentation following VSV infection. Wild type CMT.64 cells andvector transfected CMT cells infected with VSV are not recognised byCTL. FIG. 17 shows that Influenza virus is not presented to specific CTLby CMT-TAPI cells. CMT.64, RMA and CMTr1.4 cells were infected withInfluenza virus and exposed to influenza-specific CTL. In this casehowever there was no recognition of the CMT-TAP-1 cells. Both positivecontrols. RMA and CMT.64+Inf, were efficiently lysed by the CTL.

Additional experiments were carried out showing that VSV expressionrequires only the expression of the TAP-1 transporter, and thatrecognition of CMT.64 cells expressing the rat TAP-1 gene alone isalmost as efficient 20 as cells expressing both transporters, TAP 1 andTAP-2. Expression of TAP-2 alone did not appear to be as efficient (FIG.14). Different rat TAP-1 clones were also analyzed and confirmed theprevious conclusions (FIG. 15).

Influenza virus infected cells were found to be efficiently recognizedonly if both rat TAP genes are present (FIGS. 16 to 18). FIG. 16 showsthat RMA cells and CMT.64 cells treated with IFN-γ are recognizedefficiently after influenza infection. CMT.64 cells 30 transfected withthe rat TAP-1 gene are not recognized. FIG. 17 shows the same resultsobserved in a second independent experiment. FIG. 18 shows that inaddition to RMA cells and CMT.64 cells treated with IFN-γ. CMT.64 cellstransfected with both rat TAP genes are recognized after influenza virusinfection. FIG. 19 shows that HSV infected cells are recognized byspecific CTL independently of the expression of the rat TAP-1 and/orTAP-2 transporter genes.

These results provide evidence that an individual TAP-1 transportermolecule can restore the antigen presentation capability to a deficientcell in the absence of TAP-2. This finding correlates with the recentobservation that the TAP-1 protein interacts with MHC class I heavychain in cells that do not express TAP-2 (Suh, W-K., et al. Science264:1322-1326, 1994). This calls into question the absolute requirementfor heterodimer formation between the two putative transporter moleculesand demonstrates that different forms of transporter complex arefunctional and mediate transport of distinct subsets of the antigenicpeptide pool for assembly with MHC class I molecules.

Example 13 Effects of TAP on Tumor Survival

Mice were injected with CMT-64 cells or CMT.12.12 cells (a rat TAP-1 andTAP-2 transfected clone) ip at 2×10⁵ and 5×10⁵ cells per mouse. The celllines were resuspended in PBS prior to inoculation into recipient mice.The results are shown in Table 2. One of the mice treated with 5×10⁵CMT.64 cells was sacrificed and an autopsy clearly revealed the presenceof a solid tumor at the site of injection. Furthermore, all mesentericlymph nodes were grossly enlarged.

Example 14

The following is an experimental approach for finding peptidestranslocated in the ER by TAP-1 transporters, TAP-2 transporters andTAP1-TAP-2 transporters.

1. Use of cell lines expressing TAP-1. TAP-2 and TAP-1 TAP-2transporters; for example CMT.64 cells transfected with cDNA from TAP-1and TAP-2, CMT.64, CMT.14, CMT.2-10, CMT.12-12.2. Subcellular fractionation of these cells.3. Isolation of the ER (endoplasmic reticulum).4. Extraction of the peptides from the ER in 0.1% TFA.5. Gel filtration.

6. Reverse-Phase HPLC.

7. Fractions can be collected and tested for CTL sensitization or forradioactivity if cells were labelled.8. Finally, sequenced for amino acid.

Comparison of HPLC profile from different cell lines expressingdifferent TAP transporters will provide information about peptidetransport dependency on TAP molecules. Peaks of peptide can be isolatedand analyzed. Sequencing of the peptides will provide information on themotif necessary for transport using TAP-1 alone, TAP-2 alone or TAP-1and TAP-2 molecules. The protocol for peptides analysis and sequencingis standard and described in Rammensee papers and in Engelhard's papers.

Example 16 Anti-Sense Knockout in RMA-S Cells

This preliminary experiment was carried out on bulk selected populationsof cells. The construct used was the same pHβA-neo that all the MTP 1and 2 clones were obtained with as described above. In this study theMTP1 cDNA insert was cut out and it was replaced with a sequence in theopposite orientation. RMA-S cells only have TAP-1 not TAP-2, so only theMTP1 antisense was used. The data below are from single-colour FACSanalysis, the numbers are linear (5000 events counted/sample). The28.14.8s antibody is specific for D^(b), and the Y-3 antibody isspecific for K^(b). The antisense construct is designated RMA-S.ptml.

CELL ANTIBODY MEAN FLUORESCENCE RMA-S — 56.6 RMA-S 28.14.8s 129.9 RMA-SY-3 222.2 RMA-S.ptml — 53.4 RMA-S.ptml 28.14.85 96.7 RMA-S.ptml Y-3148.8

Example 17 Effect of TAP-1 and TAP-2 on Survival of Mice Injected withTumor Cells

The survival of syngeneic C57BL/6 and control allogeneic Balb/C miceinjected with very high doses of CMT.64 cells (from C57BL/6 mice) orCMT.12.12 cells (CMT.64 cells transfected with TAP-1 and TAP-2) wasinvestigated as follows.

5×10⁵ CMT.64 or CMT.12-12 cells were injected into the miceintraperitoneally and the mice were followed for 90 days. Mice wereautopsied after death or after 90 days. Survival of the mice is shown inFIG. 20. Results of the autopsies are summarized in Table 3. All of thesyngeneic C57BL/6 mice injected with CMT.64 cells were dead before 60days. These mice were found to have invasive generalized metastasizedtumors throughout the body, and exhibited ruptured organs and/orperforated intestines with excessive fluid in the peritoneal cavity.This was classed as type B pathology. Approximately 20% of the syngeneicmice injected with CMT.12.12 cells were alive at 60 days and 3 out of 20were alive at 90 days. Seventeen of these mice out of a total 20 hadtype B pathology two had no apparent pathology and one had type Apathology, described below.

Approximately 70% of the allogeneic control mice injected with CMT.64cells were alive at 90 days exhibiting no significant pathology. The fewmice which did exhibit any pathology had only small tumors (4-15 mm) atthe site of the injection. This was classed as type A pathology. 100% ofthe allogeneic mice injected with CMT.12.12 cells were alive at 90 days,exhibiting no pathology.

The results show that syngeneic mice injected with CMT.12.12 cellssurvived longer than those transfected with CMT.64 cells, probably dueto improved MHC Class I antigen presentation and recognition by the hostimmune system. The syngeneic mice transfected with CMT.12.12 survivingat 90 days showed no or little pathology.

Example 18 Materials and Methods Animals

The mouse strains C57BL/6 (H-2^(b)), and Balb/C(H-2^(u)). were obtainedfrom Jackson Laboratories. The mice were maintained according to theguidelines of the Canadian Council on Animal Care. The mice used in theexperiments were between 6 and 12 weeks of age.

Plasmids and Bacterial Strains

The plasmid pJS5 (a generous gift from Dr. B. Moss, NIH, Bethesda, USA)was a shuttle vector used first in bacteria to clone in the gene forrTAP1, or the minigene for amino acids 52-59 of the VSV N protein (VSVNP). The minigene included a methionine start site in front of the 8amino acid coding sequence, as well as a translational stop codon at theend. DNA sequencing verified that the correct sequence and orientationof the minigene in pJS5 were correct. The pJS5 plasmid containing thecloned gene was then transfected into mammalian cells for homologousrecombination with wild type Vaccinia Virus (VV). The pJS5 plasmidcontains an E. coli ampicillin resistance gene for plasmid selection inbacteria, as well as an E. coli guanine phosphoribosyl transferase (gpt)resistance gene for selection of recombinant VV (rVV) in cells. pJS5contains two synthetic VV promoters in front of a multiple cloning site(MCS) where either rTAP1 or VSV NP were cloned into, giving the plasmidpJS5-VSV NP. The entire section including the two promoters. MCS, andgpt resistance gene is flanked by the 5′ end of a thymidine kinase (tk)gene downstream, and the 3′ end of the tk gene upstream, for homologousrecombination into the tk gene of VV. The plasmids were amplified in theE. coli strain DH5aF′ grown in Luria-Bertani (LB) medium or on LBagarose plates containing 50 μg/ml ampicillin.

Oligonucleotides

The two complementary oligonucleotides used to make the minigene for VSVN52-59 were synthesized using an Applied Biosystems automated DNAsynthesizer model 380A at the NAPS unit (U.B.C., Vancouver, Canada). Theoligonucleotides and the amino acids they coded for were as follows:

(SEQ ID NO: 1) SacI overhang                                    1)3′-TCGAG-TAC-TCT-CCT-ATA-CAG-ATG-GTT-       NheI overhangCCG-GAG-ACT-CGATC-P (SEQ ID NO: 2) 2)5′-P-C-ATG-AGA-GGA-TAT-GTC-TAC-CAA-GGC- CTC-TGA-G (SEQ ID NO: 3) Peptidestart-arg-gly-tyr-val-tyr-gly-gly-leu-stop P = PO3

After purification on Pharmacia nick spin columns, equimolar amounts ofeach oligonucleotide were annealed in ligase buffer (BoehringerMannheim) for 2 minutes at 95° C. before cooling slowly to roomtemperature. The oligonucleotides were designed to provide overhangscorresponding to cleaved restriction sites for direct ligation into theNhel and Sacl cut pJS5 vector.

Viruses

The rVV were made by transfecting the plasmids into the VV infectedB-SC-1 cell line where the wild type VV and transfected plasmidsunderwent homologous recombination at the thymidine kinase (tk) gene ofVV. Once the rVV were isolated by selection with XMH, and recombinationwas verified by Southern blotting, large rVV cultures were grown forlater purification of the rVV on sucrose gradients. Purification of theNV from the cellular debris was considered essential in order toeliminate any immunological responses by the mice to cellular materialfrom Hela. CV-1, or BS-C-1 cells. Crude cell viral lysates were used forinfecting cells in vitro. VSV stocks were grown on Vero cells in DMEMmedium containing 10% FBS, P/S, and aliquots of the culture supernatantkept at −80° C. according to (ref.). Vaccinia virus (VV) stocks weregrown on CV-1 cells for small stocks and Hela 83 cells for the largerstocks. CV-1 cells were used to titre the VV stocks. The infections ofthe cell lines by VV were generally carried out at a MOI of 5-10. The VVwas first trypsinized with 0.1 volume of 2.5 mg/ml trypsin (1:250: DifcoLaboratories Inc., Detroit, Mich.) at 37° C. for 30 minutes, vortexingevery 10 minutes, to break up the aggregated VV before being diluted ina small volume of DMEM media containing 2% FBS. This viral inoculum wasadded to cells previously washed with PBS and allowed to incubate for60-90 minutes at 37° C. in a humidified, 5% CO₂/95% air environment.Then, complete medium containing 7-10% HI FBS was added and the cellsallowed to grow until needed for an assay, or until the culturedemonstrated 95% cytopathicity. When VV infected cells were required foran assay the infection was generally performed for 4-24 hours before thecells were removed with 0.25% trypsin, or versene plus 0.05% trypsin.

Recombinant VV was constructed by homologous recombination of the wildtype VV WR strain by infecting CV-1 cells transfected with the plasmidspJS5 or pJS5-VSVNP according to previously described protocols (Macketet al., 1989).

Generation of Effector Cell Populations

Virus-specific CTL populations were generated by infecting miceintraperitoneally (ip) with 10⁷ tissue culture infection dose (TCID)units of VSV or at the suggested plaque forming units (pfu) for VV-VSVNP and VV-TAP. CTL were collected on day 5 post immunization from thecervical lymph nodes (LN), or spleen and cultured in RPMI-1640 mediumcontaining 10% HI HyClone FBS (Gibco), 20 mM Hepes, 2 mM L-glutamine,0.1 mM essential amino acids, 1 mM sodium pyruvate, 50 mMb-mercaptoethanol (b-ME), and penicillin/streptomycin (henceforthreferred to as RPMI complete medium). The LN cell suspensions werecultured at 4×10⁶ cells/ml for 3 to 5 days in the absence of stimulationbefore being used in a CTL assay, whereas the splenocyte suspension wascultured for 7 days with peptide stimulation. Bulk populations ofVSV-specific CTL were maintained by weekly restimulation with 1 μM VSV Npeptide (amino acids 52-59) plus pulsed irradiated (2200 rads)stimulator splenocytes. Irradiated stimulator cells and CTL wereincubated together at a ratio of 4:1 in RPMI complete medium containing20 units/ml hIL-2. Seven days later, this bulk population was used in aCTL assay.

Cytotoxicity Assays

Target cells for the CTL assays were loaded with ⁵¹Cr by incubating 10⁶cells with 100 μCi of ⁵¹Cr (as sodium chromate. Amersham) in 250 μl ofCTL medium (RPMI-1640 containing 10% HI FBS, 20 mM Hepes) for 1 hour.Following three washes with RPMI, 2% FBS, the target cells wereincubated with the effector cells at the indicated ratios for 4 hours.100 μl of supernatant from each well was collected and the ⁵¹Cr releasewas measured by a compugamma computer (LKB Instruments). The specific⁵¹Cr release was calculated as follows: [(experimental− mediacontrol)/(total− media control)]×100%. The total release was obtained bylysis of the cells with a 5% Triton-X 100 (BDH) solution.

Results and Discussion

The TAP complex is responsible for maintaining a supply of peptides toMHC class I molecules and it has been suggested that the supply ofpeptides may be a limiting factor in the number of stable MHC class I onthe cell surface (SUH. W. K., et al., Science 264:1322; Ortmann, B. atal. Nature 368:864). TAP retains ‘empty’ MHC class I in the ER until itbinds peptides. If increasing the expression of TAP to a cell couldincrease the number of MHC class I molecules on the cell surfacepresenting immunogenic peptides, then perhaps the inclusion of genesencoding TAP, and a gene encoding a cytotoxic epitope in VV vectorscould increase the specific antigen presentation.

A subunit vaccine was required in order to investigate whether thepresence of TAP could enhance antigen presentation in vivo, thus a modelsubunit vaccine (VV-NP) was created using VV as a carrier, and theimmunodominant cytotoxic epitope (amino acids 52-59) of the VSV Npeptide. The VSV N peptide was chosen because it is known to bind toH-2K^(b) and elicit a specific CD8+ CTL response (van Bleek, G. M. etal. Nature 348:213; Fremont, D. H. et al. Science 257:919; Kundig, T. M.et al. Proc. Natl. Acad. Sc. USA 89:7757) and is the dominantimmunogenic epitope in VSV. More than 80% of CTL are directed againstthis epitope (Rotzschke t al. Nature 348:252; Byrne et al., J. Virol.51:682; Harty, J. T. et. al. J. Exp. Med. 175:1531; Feltkamp, M. C. W.,et al. Mol. Immunol. 31:1391; Weidt, G. et al. J. Immunol. 153:2554). Amin(gene encoding the cytotoxic epitope of the VSV N protein, aminoacids 52-59, was created using oligonucleotides which were inserted intothe pJS5 plasmid to create pJS5-VSV/NP. Once the pJS5-VSV/NP plasmid wasconstructed it was used to create the rVV containing the VSV N52-59peptide (VV—NP). A rVV vector only control (VV-pJS5) was alsoconstructed.

The VV—NP was tested in vivo to see if it could elicit an anti-VSVresponse. As seen in the CTL assay in FIG. 21 the splenocytes from miceinjected with VV—NP were able to lyse RMA targets infected with VSV butnot uninfected RMA targets. The minimum amount of VV—NP required toelicit an immune response in C57BL/6 mice was 10⁴ plaque forming units(pfu) and the maximum immune response was achieved at 10⁵-10⁶ pfu. At ahigher titer of 10⁸ pfu (10⁷ and 10⁸ not shown) the response decreasedand VV levels above 10⁸ pfu were lethal. There does not appear to be anysignificant difference in the response elicited by doses of VV-NPbetween 10⁵ and 10⁷ pfu, so the median dose of 10⁶ was chosen for theremaining assays.

The splenocytes in the mice injected with VV-NP include cytotoxiclymphocytes that were specific for VSV (FIG. 22). It was also determinedin a CTL assay with VSV N52-59 pulsed targets, that the immune responseelicited in mice injected with VV-NP included CTLs that were specificfor the VSV N52-59 cytotoxic epitope (FIG. 22). The mice injected withthe control vector VV-pJS5 did not contain VSV specific lymphocytes asthey gave the same low response seen in mice injected with PBS.Splenocytes from the VV-NP primed mice were also able to recognize VVinfected RMA targets but to a much lesser degree than they recognizedthe VSV N peptide epitope.

As the TAP complex is responsible for transporting peptides across theER lumen and for binding to MHC class I, a functional assay for the TAPactivity involves the restoration of antigen presentation in a TAPdeficient cell. In order to demonstrate this, a CTL assay specific forthe VSV N52-59 cytotoxic epitope was used to verify that the TAP 1 and 2produced by VV are functional (FIG. 22).

In order to determine whether TAP gene expression is limiting in the VSVresponse, the number of VSV N peptide-specific cytotoxic lymphocytes inthe spleen was quantified. An increase in lysis of the VSV infected RMAtargets were indicated, suggesting an increase in the number of VSVspecific CTL (FIG. 23A). The in vivo immune response to VV-NP wasamplified when the mice were simultaneously injected with the VV-hTAPs.The largest increase in response over VV-NP alone was seen with VV-hTAP1and 2, and to a lesser degree with VV-hTAP1 and VV-hTAP2. This suggeststhat the addition of a TAP gene enhanced the VSV NP-specific CTLresponse. It also demonstrated that including both TAP1 and TAP2 geneswas more effective than using either TAP1 or TAP2 alone (FIG. 23A). Alimited dilution analysis (LDA) was performed to confirm that mice,which received VV-hTAP12 and VV-NP, contained more VSV specific CTL. TheLDA data demonstrates that mice that received VV-NP had 1 VSV specificpCTL for every 78,000 splenocytes, whereas mice that also receivedVV-hTAP12 had 1 pCTL for every 16,000 splenocytes (FIG. 23B). Theaddition of TAP, therefore increased the number of VSV specific CTLapproximately 5 fold. In order to directly assess whether TAP expressionwould directly augment responses against the wild-type virus, the sameexperimental protocol was attempted using a low dose of wild type VSV(2.1×10³ TCID50) along with 1.35×10⁴ pfu VV-hTAP1 and 2 (FIG. 24). Thedose of VSV used to give the minimum immune response was approximately10,000 times less than the usual dose used for VSV immunization. It isclear that immunizing with VV-hTAP1 and 2 and VSV resulted in a largeincrease in immune responsiveness against VSV. This suggests that TAPcould be a suitable candidate for increasing an immune response to lowdoses of antigen.

With the use of adjuvants, the immune response can be modulated for aMHC class I or II response. Adjuvants like immunostimulating complexes(ISCOMs), that are made of non-covalenty bound complexes of Quil A,cholesterol, and amphipathic antigen can stimulate a CD8+ CTL response(Takahashi. H. et al. Nature 344:873). Similarly, the T cellcostimulatory molecule B7 has been shown to enhance protection againstpoorly immunogenic tumours (Townsend. S. et al. Science 259:368; Chen,L. P. et al. Cell 71:1093). In addition a wide variety of cytokines havebeen used to direct responses to either a CTL mechanism or T helperresponse. For example, interleukin-2 (IL-2) and IL-12 have been used toelicit a Th1 response which is more conducive to cytotoxic mechanisms(Hughes et al. Immunol, 74:461; Flexner et al. Vaccine 8:17; Heath etal. Vaccine 10:427); Meuer et al. Lancet 1:15). One adjuvant that hasbeen widely used in animals is Freund's complete adjuvant (FCA) which isan emulsion containing heat killed mycobaterium tuberculosis. Despitethe strong antibody responses that FCA produces, it is too toxic to beused in humans. However, derivatives for the minimal structure of themycobaterium in FCA that is needed for adjuvanticity. N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), such as murabutide do not have asmany toxicity problems (Cox et al. Vaccine 15:248).

The inventors have shown that augmentation of TAP expression doesincrease the immune response to the VSV. These data surprisingly implythat TAP expression or activity is limiting in normal cell lines and isthe first component of the antigen processing pathway demonstrated to bein short supply in healthy mice. TAP is so effective that over 10,000fold less wild-type virus could be used to ilicit the equivalent immuneresponse. When designing subunit vaccines, one may want to reduce theamount of innoculum used for vaccination. This is important not only forefficiency in distributing large amounts of vaccine but is alsoimportant when larger doses of a weakly immunogenic peptide are required(Melnick, Acta Virol. 33:482; Arnon et al. Cur. Op. In Immunol 4:449).The inclusion of TAP in vaccination regiments could address theseproblems. An additional advantage of including TAP as an adjuvant isthat its ability to increase peptide transport of a number ofimmunogenic peptides simultaneously. In a virus containing a complexarray of peptides, one could envisage that the inclusion of TAP wouldincrease the delivery of most epitopes. Furthermore, this would aid inthe delivery of diverse peptide for binding to most HLA allelesexpressed in the population being immunized. TAP could be used as anadjuvant in peptide vaccines but it does not have to be restricted toviral vectors. For example, it could also be injected in other formssuch as in DNA plasmids attached to gold particles or any other systemwhich inserts the TAP complex directly into the cell's proteinprocessing pathway (Dertzbaugh Plasmid 39:100). Future clinicalexperiments will help to further establish whether the inclusion of TAPin vaccine regiments has advantages over existing protocols, whetherother components of the intracellular antigen processing pathway(s) arealso limiting in healthy individuals.

Example 19 Material and Methods Mice

C57BL/6 (H-2^(b)) mouse strains were bred at the Animal Care Centre atthe University of British Columbia. All mice used for the experimentswere 6- to 12-weeks-old and were maintained in accordance with theguidelines of the Canadian Council on Animal Care.

Generation of CTL Cultures and Cytotoxicity Tests

H-2K^(b)-restricted VSV- or Sendai virus-specific CTLs were generated byi.p. injection of either VSV or Sendai virus alone or one of theseviruses plus recombinant vaccinia virus (VV) carrying with (VV-TAP1,2)or without (VV-PJS-5) human TAP 1 and 2 genes into C57BL/6 mice with theviral dose indicated in each figure. After 5-6 days immunization, thesplenocytes were removed and cultured in RPMI-1640 complete mediumcontaining 10% heat-inactivated HyClone FBS (GIBCO BRL), L-glutaimine,100 IU/ml penicillin, 100 mg/ml streptomycin, Hepes, 0.1 mMnon-essential amino acids, 1 mM Na-pyruvate, and 50 mM 2-ME. Thesplenocyte cultures were incubated at 3×10⁶ cells/ml at 37° C. for 3days with the peptide (1 μM Sendai-Np 324-332 peptide, FAPGNYPAL) forSendai-specific effectors or without a peptide for VSV-specificeffectors. The erythrocytes were removed from the splenocytes before 3days culture (for VSV-specific effectors) or after (for Sendai-specificeffectors).

The cytotoxic activity was measured in standard 4-h ⁵¹Cr release assays.The RMA target cells were pulsed with 5-25 μM either VSV-Np (RGYVYQGL(SEQ ID NO:5)) or Sendi-Np (FAPGNYPAL (SEQ ID NO:6)) peptide forrelevant CTL response. The targets were labeled with Na⁵¹CrO4 (100μCi/10⁶ cells) for 1 hr at 37° C. and cytotoxic activity was assayed ina standard 4 h ⁵¹Cr release assay. The cytotoxicity tests were done in96 V-shaped well plates at many effector:target ratios.

Analysis of Frequency of VSV-specific CTL Precursor

The frequency of CTL precursor was detected by limiting dilutionanalysis (LDA). Briefly, the assay was performed in U-bottom 96-wellplate supplied with the complete medium containing 20 U/ml recombinantmurine IL-2, 5% supernatent of con A-stimulated rat splenocytes and 0.1M μ-methyl-D-mannoside. Wells contained graded concentrations of theimmunized splenocytes and both irradiated cells, 1×10⁵ syngenicsplenocytes as the feeders and 3×10³ VSV-Np 52-59 peptide-pulsed RMAcells as the stimulators. The cells were cultured at 37° C. for 7 daysand at day 6, 80 μl cultured medium in each well was replaced with sameamount of fresh one. On day 7, a standard CTL assay was performed byreplacing 100 μl supernatant with 5 μM VSV-Np peptide-pulsed,⁵¹Cr-labeled RMA cells in each well as targets. Kinetic analysis and CTLprecursor (CTLp) frequency determinations were performed by thestatistical methods of X² minimization as described by Taswell [Taswell,1981 #1].

Detection of TAP Expression and Activities

Human TAP1 expression in immunized mouse splenocytes were determined byimmunoblotting. Total extracts from 1×10⁵ cells were separated on 10%polyacrylamide-SDS gels and blotted onto nitrocellulose filters. Theblots were probed with TAP C-terminus-specific rabbit antisera (giftsfrom Dr. Monaco, J. J.) at a 1:1000 dilution for anti-human TAP1. Theblots were then incubated with horseradish peroxidase-labelledanti-rabbit antibodies at a 1:10,000 dilution. The immunocomplexes werevisualized by enhanced chemiluminescence (ECL) according to theinstructions of the manufacturer (Amersham, UK). Tne naive mousesplenocytes were used as negetive controls.

TAP heterodimer activities were detected by streptolysin-O mediatedpeptide transport assays as described by Androlewicz et al.[Androlewicz, 1993 #2] with minor modifications. Briefly, apeptide-library which contains 3240 different peptides with aglycosylation site (NXT) in each was labeled with ¹²⁵I by chloramineT-catalyzed iodination to a specific activity of 10 Ci/mmol. 2×10⁶splenocytes from naive, TAP−/−. VV-PJS-5- or VV-TAP1,2-immunized micewere permeabilized with 2 IU/ml streptolysin-O (Murex, Norcross, Ga.)for 15 min at 4° C. After removing unbound streptolysin-O and the cellswere resuspended in 37° C. intracellular transport buffer (50 mM Hepes,pH 7.0, 78 mM KCl, 4 mM MgCl2, 8.37 mM CaCl2, 1 mM EGTA, 1 mMdithiothreitol (DTT). Adjust pH to 7.3 with KOH) for 5 min to initiatepore formation. The iodinated peptide-library (˜66 ng) was then addedimmediately. The incubation was continued for another 10 min in thepresence or absence of 10 mM ATP (Sigma Chemical Co., St. Louis, Mo.).Afterwards, the cells were transferred to ice and were lysed in a buffercontaining 1% NP40, 150 mM NaCl, 5 mM MgCl₂, 50 mM Tris-HCl pH 7.5. Thenuclei were removed by centrifugation of samples at 14,000 rpm for 10min. Translocated peptides that had been glycosylated in the ER wererecovered by absorption to concanavalin A-Sepharose beads (PharmaciaDiagnostics, AB). The beads were washed five times in lysis buffer. Theassociated radioactivity was measured in a γ-counter (model 1282CS; LKBPharmacia).

Example 20 TAP1 Increases the Expression of MHC Class I on the Surfaceof a Metastatic Prostate Cancer Cell Line Background

The inventors have examined the effect of TAP1 gene transfection on thelevel of MHC Class I displayed by cells derived from a metastatic cancermodel developed by the laboratory of Timothy C. Thompson from the BaylorCollege of Medicine, Houston. Tex. (1). This model of metastatic diseasemakes use of a primary prostate cancer cell line 148-1 PA and ametastatic cell line 148-1 LMD derived from the same clone. Theparental, primary cell line has been shown to be more immunogenic thanthe metastatic 148-1 LMD line as examined by the growth of the tumor inimmune competent, syngeneic mice. Interestingly, while both cell lineswere capable of inducing syngeneic and allogeneic antitumor CTL, onlythe parental cell line was susceptible to killing by antitumor CTL. Bothtumors, however, express reduced levels of genes involved in antigenpresentation such as: TAP1, TAP2, LMP-2 and LMP-7 (24). It is possiblethat the expression of these genes is not sufficient for effectivepresentation to specific CTL as is the case for the metastaticderivative of this prostate tumor model.

Materials and Methods

The cell lines described by Lee et al. (24) were maintained in DMEM and10% Fetal Calf Serum (FCS) at 37 C and 5% CO₂. Human TAP1 cloned in themammalian expression vector pCEP4 (Invitrogen) was obtained withpermission of use from Ping Wang, University of Lund, Sweden. Vectorcarrying human TAP1 (10 μg) was incubated with Lipofectamine reagent (8μl) (Life Technologies) in 200 μl of serum-free media for 15 min. Thenthe mixture was overlayed on 4×10⁵ metastatic 148-1 LMD in a total of 1ml of serum-free media and incubated 4 hr further. Then FCS was added toa final of 10% concentration and the cells were incubated for anadditional 48 hr. The selection of stable transfectants was carried outby incubating the cells in 200 μg/ml hygromycin with regular passage forone month.

Fluorescence Activated Cell Sorting (FAGS) machine (BD-FACScalibur) wasused to measure the expression of MHC class I expression on the cellsurface. Clones were isolated and examined for their expression of MHCClass I by staining the cells with Y3 followed by washing and stainingwith FITC conjugated goat anti-mouse IgG. Y3 anti H2-Kb antisera is aconformational dependent antisera that binds to MHC class I only in thepresence of bound antigenic peptide. As a control for non-specificfluorescence the primary antibody was omitted from the stainingprotocol.

Results and Discussion

The examination revealed a severely impaired expression of MHC Class Ion the cell surface in both the metastatic and primary cell lines. Theinventors were able to reconstitute the expression of MHC Class I on thecell surface by transfecting these cells with TAP1 (FIG. 30A) or, in theabsence of TAP1, by treating the cells with interferon-γ (FIG. 30B).

FIG. 21 TAP1 Augmenting B16F10 Tumor Antigenicity And ImmunogenicityBackground

B16F10 is a well-studied melanoma cell line derived from C57BL/6 mice.The cell line expresses the tumor-associated antigens TRP-1. TRP-2, andgp 100. B16F10 is not immunogenic and is resistant in vivo to IL-2treatment and adoptive transfer of CTL. In vivo, immune responses arenot elicited even when the cells are transfected with the accessorymolecule B7.1 or when mice are vaccinated with cells mixed with BCG.

The non-immunogenicity of B16 F10 can be attributed to defects in MHCclass I restricted antigen presentation. MHC class I, TAP1, TAP2,tapasin, proteasome LMP2. LMP7 and PA28 are down regulated in this cellline (25).

The inventors show that transfection and expression of TAP1 alone,restores the presentation of melanoma tumor associated antigen (TRP-2)in the context of MHC class I, despite the multiple defects in theantigen presentation pathway. In addition to the TRP-2 antigen, TAP1transfection of B16F10 cells also facilitates the presentation of theviral antigen, VSV-NP.

The increase in MHC class I antigen presentation is sufficient for bothtumor and viral antigens in the context of MHC I to makes TAP1transfected B16 F10 susceptible to killing by TRP-2 specific and\/8V-NP, cytotoxic T-cells respectively. Also, mice bearingpre-existing, non-immunogenic B16 F10 tumors generate specificanti-tumor immune responses when treated in vivo with a vectorcontaining TAP1.

Materials and Methods Transfection

The transfection of melanoma cell line B16 F10 with rat TAP1 and netTAP2 followed the protocol outlined in Materials and Methods forExamples 1-8.

Defection of MHC class I molecules

Surface expression of the H-2K^(b) allele was detected by indirectimmunofluorescence using the conformational-dependent mouse monoclonalantibodies (mAbs), Y-3 (ATCC), which is specific for K^(b)-b₂Mcomplexes. Fluorescein isothiocynate-conjugated (FITC) rabbit anti-mouseIgG (Dakopatts, DK) was used as the secondary antibody. A FACScananalyzer (Becton and Dickinson, Mountain View, Calif.) measured the meanlogarithmic fluorescence intensity.

Detection of rat TAP genes

RT-PCR analysis of rat TAP mRNA expression was performed in B16F10TAP-transfectants. Total RNA was extracted by using the RNeasy Kit(Qiagen) according to manufacturer's protocol. Random-primed cDNA wasgenerated using the RETROscript, RT-PCR Kit (Ambion) following themanufacturer's instructions. The inventors then used 0.5 μg of cDNA fromeach spleen to amplify sequences corresponding to rat TAP1 and TAP2using the following primer sets: GACCGGACTCTGGACAGC andGTAAATTCCGGGGCATCTCCT corresponding to rat TAP1: AGGAAGCAGATTTCAGAACTCand AGTCCTGAGAGGGCTCAGTGT corresponding to rat TAP2 respectively. Theβ-actin subunit primer set was obtained from Ambion. For all targets,the PCR reaction consisted of 30 cycles of amplification at an annealingtemperature of 56° C. using Platinum Taq polymerase (Invitrogen),according to manufacturer's instructions. One tenth of the product ofeach PCR reaction was examined by agarose gel electrophoresis. Theinventors measured the intensity of β-actin product in order to ensurethat the reaction kinetics and starting material of cDNA in eachreaction was equivalent.

Generation of Effector Cell Populations

Virus-specific CTL populations were generated by infecting miceintraperitoneally (ip) with 10⁷ tissue culture infection dose (TCID)units of VSV. CTL were collected on day 5post immunization fromimmunized spleen and stimulated with 1 μM VSV-NP peptide (amino acids52-59) in RPMI-1640 medium containing 10% HI HyClone FBS (Gibco), 20 mMHepes, 2 mM L-glutamine, 0.1 mM essential amino acids, 1 mM sodiumpyruvate, 50 μM mercaptoethanol (ME), and penicillin/streptomycin(henceforth referred to as RPMI complete medium). Seven days later, thisbulk population was used in a CTL assay.

For tyrosinase-related protein 2 (TRP-2) specific CTL generation, thespecificity of splenocytes was generated by injecting miceintraperitoneal with 3×10⁶ γ-irradiated RMA-S cells pulsed with 5 μM,K^(b)-restricted TRP-2 peptides (SVYDFFVWL) for 5 days. Upon removal thesplenocytes were cultured with 1 μM TRP-2 for 6 days and used forbulk-culture CTLs in a standard 4 h ⁵¹Cr release assay.

Cytotoxicity Assays

Target cells (B16F10 and rat TAP1-transfectant. B16.TAP1) for the CTLassays were loaded with ⁵¹Cr by incubating 10⁶ cells with 100 μCi of⁵¹Cr (as sodium chromate, Amersham) in 250 μCTL of CTL medium (RPMI-1640containing 10% HI FBS, 20 mM Hepes) for 1 hour. Following three washeswith RPMI, 2% FBS, the target cells were incubated with the effectorcells at the indicated ratios for 4 hours. 100 μl of supernatant fromeach well was collected and the ⁵¹Cr release was measured by a O-counter(LKB Instruments). The specific ⁵¹Cr release was calculated as follows:[(experimental− media control)/(total−media control)]×100%. The totalrelease was obtained by lysis of the cells with a 5% Triton-X 100 (BDH)solution.

Inoculation of Mice with Tumor Cell Lines and Tumor-Therapy

1.5×10⁵ B16F10 cells in PBS were injected subcutaneously (s.c.) intoC57B1/6 syngeneic mice. One day after, the mice received s.c. 1×10⁶(PFU) of either vaccinia vector alone (VV-PJS-5, 5 mice per group) orvaccinia-carrying rat TAP1 (VV-rTAP1, 5 mice per group). This procedurewas repeated 7 days later. All mice were killed 17 days after theintroduction of the tumor cells. The tumor sizes are measured and thedifference between two groups was analysed by a one tailed Student'st-text (p<0.5).

Results and Discussion

It has been reported that the murine melanoma cell line. B16F10,down-regulates the expression of MHC class I molecules and TAP1 and TAP2proteins (25). These defects result in cells unable to present tumorantigen for recognition by immune system. In a mouse small cell lungcarcinoma cell line. CMT.64, that shows a phenotype similar to B16F10,the inventors transfected rTAP1 gene into the cells and found that thetransfectants increased its antigen presentation capacity (26). To seewhether the same treatment can also apply to B16F10 cell line, theinventors transfected rat TAP1 and/or TAP2 gene in to the B16F10 cellline. Transfection was confirmed by RT-PCR and all transfectants expressthe introduced rTAPs (see FIG. 31A,B). B16F10 transfected with rTAP1expresses higher surface H-2K^(b) molecules than wild-type andvector-transfected cells (FIG. 32) demonstrating that TAP is essentialfor antigen presentation despite other deficiencies in the antigenpresentation pathway.

To see if introducing rat TAP1 gene into B16F10 cells restores theantigenicity and immunogenicity of these tumor cells, the inventorsperformed a cytotoxicity assay to test the cells' capacity on thepresentation of K^(b)-restricted epitopes derived from VSV. Thesusceptibility to specific lysis of TAP transfected B16F10 cells wascompared to untransfected B16 F10 cells and B16 F10 cells transfectedwith vector alone. Each of these 3 cell lines was infected with VSV(1:10 moi) and cell lysis by antigen specific splenocytes was measuredwith a CTL assay. Cells pulsed with VSV-NP 52-59 peptide acted as apositive control and untreated cells were used as a negative control.The CTL assays demonstrated that only TAP transfected B16 F10 cellsinfected with VSV were lysed by VSV antigen specific splenocytes (FIG.33). Untransfected B16 F10 and B16 F10 cells transfected with vectoralone were not lysed by VSV specific splenocytes. The results of the CTLassay shows that antigen presentation and immunogenicity issignificantly increased in TAP transfected B16 F10 cells compared towild type or vector transfected B16 F10 cells. Only cells transfectedwith rTAP1 gene are able to present VSV-NP epitope significantly (FIG.33).

Since B16F10 cell line express tumor-associated antigens, TRP1. TRP2 andgp100. It is important to test whether TAP1-transfected B16F10 can alsopresent the tumor-associated antigen TRP-2, one of thesetumor-associated antigens. The TRP-2-specific CTLs were generated by thesplenocytes of the mice injected with the synthetic epitope peptidepulsed RMA-S cells. The lysis of TAP1-transfected B16F10 cells(B16.TAP1) by splenocytes specific for TRP-2 antigen is dramaticallyincreased over untransfected B16 F10 cells as demonstrated by CTL assay(FIG. 34). This result indicates that TAP1 enhanced the presentation ofan endogenous tumor-associated antigen in B16F10 cells.

Next, the inventors investigated the ability of TAP1 to act as atherapeutic by in mice bearing B16 melanoma tumors. Wild-type B16 F10cells were introduced into the flanks of mice causing tumors to grow. Avaccinia vector containing the TAP1 gene was injected subcutaneous onthe same flank as the tumor on two occasions separated by seven days.The mice were killed 17 days after the tumors were introduced and thetumor mass was measured. Analysis (Student's t-test, p<0.5) of tumormass demonstrated that mice receiving the vaccinia TAP vector (VV-rTAP1)had significantly smaller tumors than mice that received a vacciniavector not containing TAP (VV-PJS-5 (vector alone)) (FIG. 35).

In conclusion increased TAP1 expression increases the amount of MHCclass I bearing both exogenous and endogenous antigen on the surface ofB16 F10 cells making them antigenic and immunogenic. This resultantincrease in immunogenicity is capable of inhibiting B16 tumor growth invivo.

Example 22 TAP Expression Improves Immune Recognition of and ProtectionAgainst Malignant Cells In Vivo Background

Neoplastic cells arise frequently in the body due to a variety ofexternal and internal influences and the inventors depend on the immunesystem to recognize and destroy these cells before they develop intotumors. However, malignant transformations may be accompanied byphenotypic changes resulting in the ability of the cancer cells toescape the immunosurveillance mechanism. As the phenotypic changes varywith each neoplasm the inventors are unable to develop one treatment forall cancers. Fortunately, many tumors fall into one of several largergroups of phenotypes. One such group presents with increasedtumorigenicity due to a decrease in MHC class I expression (27-31). Adecrease in cell surface expression of MHC class I can be due to adefect anywhere in the MHC class I biosynthetic pathway (30, 32). Thereare many cellular proteins that contribute to MHC class I assembly(33-36). The TAP complex is one of the most important components. Thefunction of the TAP complex is to supply endogenously-processed peptidesfrom the cytosol into the ER for binding to relevant MHC class I,resulting in cell surface presentation of these complexes to CTLs. Lossof the TAP complex is highly correlated with loss of HLA expression incervical carcinoma (37). In addition, a higher frequency ofdownregulation of this complex has been observed for metastatic lesionsthan for primary lesions (29). Particularly, the TAP complex has beenimplicated in tumorigenicity of several cancers such as melanomas,cervical carcinomas, and renal cell carcinomas (29, 38, 39). Thesefindings suggest that TAP downregulation may represent an importantmechanism for immune escape of malignant cells in a variety of tumors.

The immune system has evolved a very intricate recognition mechanism toeliminate diseased cells. In order for a tumor to proliferate it has toevade the cells involved in tumor recognition. The major anti-tumoreffector mechanisms are the CD8⁺ CTLs and NK cells (reviewed in 30). Thefunctions of both effectors are controlled by MHC class I. CD8⁺ CTLsrecognize surface MHC class I restricted tumor associated antigens (TAA)and destroy tumor cells (40), whereas NK cells only lyse the targetswith absence or downregulation of surface MHC class I.

Small cell lung carcinomas (SCLCs) are highly malignant in humans andare generally fatal. SCLCs in mice have similar characteristics. TheCMT.64 cell line is one of the SCLCs which arose spontaneously in aC57Bl/6 mouse (41). This cell line is lacking in both MHC class Isurface expression and endogenous antigen presentation. IFN-γ treatmentcorrects these deficiencies. However, the underlying defect remainsunknown (42, 43). The inventors have recently shown that CMT.64 cellshave many defects of components in antigen presentation pathway, such asMHC class I heavy chain, β₂-m, proteasome subunits (LMP 2 and LMP 7),and TAP-1 and -2 (44). These defects can be rescued by INF-γ treatment(18). Although the CMT.64 cells are very poor at antigen presentation,reconstitution with rTAP1 and rTAP1,2 restores viral antigenpresentation in vitro (44, 45). These data demonstrated that theblockage of antigen presentation in the SCLC CMT.64 is at the level oftransport of peptide from the cytoplasm to the ER.

It is difficult to treat cancers since many of them have multiplecellular defects. However, if restoring one component will allow thehosts' immune system to recognize and destroy the cancer, then thecourse of treatment would be less complicated. Many of the tumorslacking surface MHC class I have TAP losses or dysfunctions (29). IfCMT.64 transfected with TAP is sufficient for restoring antigenpresentation in vitro, does it follow that the immune system recognizethe TAP-expressing tumor cells, destroy them and thus prevent metastasisin vivo? To examine this possibility, the TAP-deficient CMT.64 as wellas TAP-transfected cell lines derived from CMT.64, were tested to see ifTAP could improve the immune response against cancer cells and thusimprove survival of animals bearing this tumor.

Materials and Methods Animals

The mouse strain C57BL/6 (H-2^(b)) was obtained from JacksonLaboratories but housed at the Biotechnology Breeding Facility(University of B.C.). The H-2^(b) nude mice {B/6 Nu-M (C57Bl/6NTAC-NufDF)} were obtained from Taconic (Hanover, N.Y., USA) and kept inspecific pathogen free incubators. The mice were maintained according tothe guidelines of the Canadian Council on Animal Care. The mice used inthe experiments were between 6 and 12 weeks of age and were sacrificedby CO₂ asphyxiation.

Recombinant Vaccinia Virus Construction (VV)

Recombinant VV was constructed by homologous recombination of the wildtype VV VVR strain by infecting CV-1 cells transfected with the plasmidspJS5, pJS5-rTAP1, pJS5-rTAP2 or pJS5-rTAP1,2 according to previouslydescribed protocols (Macket et al., 1989).

Purification of VV Stocks

Crude cell stocks were used for the infection of cells in culturehowever purified stocks of VV were used when injecting mice. To purifythe VV, 3 L batches of VV infected Hela S3 cultures were used. The VVwas released from the cells by homogenization with a Dounce Homogenizerbefore centrifugation at 750×g for 5 minutes at 4° C. The supernatantwas trypsinized with 0.1 vol. of 2.5 mg/ml trypsin for 30 minutes at 37°C., then layered onto an equal volume of 36% sucrose in 10 mM Tris-HClpH 9.0. It was centrifuged for 80 minutes at 4° C. at 25,000×g and thepellet was then resuspended in 1 mM Tris-HCl pH 9.0. The pellet wastrypsinized again before being layered onto a 24-40% continuous sucrosegradient and centrifuged for 45 minutes, at 4° C. at 18,750×g. The milkyband was collected and saved while the pellet was trypsinized andrepurified on another sucrose gradient. All of the bands collected werepelleted by diluting with 2 volumes of 1 mM Tris-HCl pH 9.0 andcentrifuging for 60 minutes at 4° C. at 25,000×g. The viral pellet wasresuspended in 1 mM Tris-HCl pH 9.0, and 0.5 ml aliquots were stored at−80° C. or −135° C.

Tissue Culture

The small cell lung carcinoma cell line. CMT.64, used in the cancerexperiments originated spontaneously from the C57BL/6 mouse strain (41).All of the stable CMT.64 transfectants containing rTAP-1 (CMT.1-4,CMT.1-10), rTAP-2 (CMT.2-1, CMT.2-10), rTAP1,2 (CMT.12-21) and thevector only control (CMT.neo) were created by transfecting CMT.64 cellswith the rTAP cDNA in mammalian expression vector pH(Apr-1neo) (44, 45).All cell lines including RMA cell line were grown in either DMEM or RPMIcontaining 10% fetal bovine serum (FBS).

Generation of Effector Cell Populations

Virus-specific CTL populations were generated by infecting miceintraperitoneally (ip) with 10⁷ tissue culture infection dose (TCID)units of VSV or at the suggested plaque forming units (pfu) for VV-TAPor VV-pJS5 vector. CTL were collected on day 5 post immunization fromthe cervical lymph nodes (LN), or spleen and cultured in RPMI-1640medium containing 10% HI HyClone FBS (Gibco), 20 mM Hepes, 2 mML-glutamine, 0.1 mM essential amino acids, 1 mM sodium pyruvate, 50 μMβ-mercaptoethanol (β-ME), and penicillin/streptomycin (henceforthreferred to as RPMI complete medium). The LN cell suspensions werecultured at 4×10⁶ cells/ml for 3 to days in the absence of stimulationbefore being used in a CTL assay, whereas the splenocyte suspension wascultured for 7 days with peptide stimulation. Bulk populations ofVSV-specific CTL were maintained by weekly restimulation with 1 (M VSV Npeptide (amino acids 52-59) plus pulsed irradiated (2200 Rads)stimulator splenocytes. Irradiated stimulator cells and CTL wereincubated together at a ratio of 4:1 in RPMI complete medium containing20 units/ml hIL-2. Seven days later, this bulk population was used in aCTL assay.

For anti-tumor CTL generation, the specificity of splenocytes wasgenerated by injecting mice intraperitoneally with 1×10⁷ CMT.neo orCMT.1-4 cells (FIG. 37C). Upon removal the splenocytes were culturedwith stimulators at a 3:1 ratio. The stimulators were prepared byincubating CMT1-4 or CMT.neo cells with 30 mg/ml mitomycin C underhypoxic conditions. After incubation of 2 hours the cells wereγ-irradiated (10,000 Rads) and washed three times before addition to thesplenocyte culture. CMT.neo splenocytes received CMT.neo stimulators,whereas CMT1-4 splenocytes received CMT1-4 stimulators. Six days afterin vitro stimulation the splenocytes were tested in a standard 4 hour⁵¹Cr release assay.

For anti-tumor and VV CTL generation, the specificity of splenocytes wasgenerated by injecting mice intraperitoneally with 1×10⁷ CMT.neo cellsand 1×10⁶ pfu VV-rTAP1 (FIG. 40B). The splenocytes were a secondary massculture which were incubated with stimulator cells, plus γ-irradiated(5,000 Rads) naive syngenic splenocytes, at a 5:1:15 ratio. Thestimulator cells were prepared by infecting CMT.neo cells with VV-rTAP1for 3 hours before adding 30 mg/ml mitomycin C under hypoxic conditions.After incubation of 2 hours the cells were γ-irradiated (10,000 Rads)and washed three times before addition to the splenocyte culture. Afterincubation of six days the splenocytes were tested in a standard 4 hour⁵¹Cr release assay.

Cytotoxicity Assays

Target cells for the CTL assays were loaded with ⁵¹Cr by incubating 10⁶cells with 100 (Ci of ⁵¹Cr (as sodium chromate. Amersham) in 250 (I ofCTL medium (RPMI-1640 containing 10% HI FBS, 20 mM Hepes) for 1 hour.Following three washes with RPMI, 2% FBS, the target cells wereincubated with the effector cells at the indicated ratios for 4 hours.100 (I of supernatant from each well was collected and the ⁵¹Cr releasewas measured by a γ-counter (LKB Instruments). The specific ⁵¹Cr releasewas calculated as follows: [(experimental−media control)/(total−mediacontrol)]×100%. The total release was obtained by lysis of the cellswith a 5% Triton-X 100 (BDH) solution.

FACS assays

Surface expression of the H-2K^(b) allele was detected by indirectimmunofluorescence using the conformational-dependent mouse monoclonalantibodies (mAbs), AF6-88-5.3 (ATCC) and 142.23 (a gift from Dr. KvistS.), both specific for K^(b)-β₂M complexes. Fluoresceinisothiocynate-conjugated (FITC) rabbit anti-mouse IgG (Dakopatts, DK)was used as the secondary antibody. The mean logarithmic fluorescenceintensity was measured by a FACScan analyzer (Becton and Dickinson,Mountain View, Calif.). Detection of CD4⁺ T-cells and CD8⁺ T-cells wasused similar protocol as detection of surface MHC class I molecules withminor modification. Briefly, tumors were washed extensively andhomogenized into single cells. FITC-conjugated rabbit anti-mouseantibodies (PharMinGen), RM4-5 (against CD4) and 53-6.7 (against CD8)was used.

Western Blots

The proteins of lysates from 5×10⁵ cells were separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a10% resolving gel and then were transferred to a nitrocellulosemembrane. The blots were probed with either the rabbit anti-rat TAP1(D90) or TAP2(114/2) polyclonal antibody at a dilution of 1:1000 andthen incubated with horseradish peroxidase-labelled anti-rabbit antibodyat a 1:100,000 dilution. The immunocomplexes were visualized by enhancedchemiluminescence (ECL) according to the instructions of themanufacturer (Amersham. UK) and were quantitatively assessed by adensitometry scan.

Inoculation of Mice with Tumor Cell Lines

5×10⁵ (otherwise indicated in figure legend) CMT.64 or its transfectantsin PBS were injected ip. into C57B1/6 syngeneic mice. For examination oftumor-growth pattern, one representative mouse from each group (4 micefor each group) was sacrificed after one month and photographing in vivotumors (FIG. 37A). For mice survival experiments, each group contained15 (FIG. 37B) or 10 (FIGS. 39B and 40A) mice.

Statistics

The statistics for the cancer studies were performed using theKaplan-Meier log rank survival test or Regression log percentagesurvival test prior to carry out a paired t-test. The computer softwareprogram JMP IN version 3.2.1 (SAS Institute Inc. (1989-97, DuxburyPress, NC. USA). was used to do the computations. The data wasconsidered statistically different if p<0.05.

Results and Discussion Phenotype of TAP Transfectants

The phenotype of the CMT.64 cell line has been previously described(44). Transfection of rat-TAPs (rTAP) into this cell line partiallyrestores expression of relevant components in MHC class I-restrictedantigen presentation pathway. FIG. 36A shows transfectants expressingrTAP proteins. Two rTAP1-transfectant clones. CMT.1-4 and CMT.1-10, andone rTAP1,2-transfectant. CMT.12-21, express higher levels of rTAP1protein than the CMT.1-1 clone (one of rTAP1 clone). In comparison withRMA cells, these transfectants reveal a similar rTAP1 expression level(except CMT.1-1): CMT.1-1 express 10-times less, CMT.1-4 and CMT.1-10express 2.5-times less, and CMT.12-21 expresses 2-times less. The rTAP2expression levels in the transfectants were also examined in relation toRMA: CMT.2-1 and CMT.2-10 (two rTAP2 transfectant clones) express4-times and 2-times less respectively, and CMT.12-21 2-times less. Thus,in comparison to RMA all rTAP transfectants, except CMT.1-1, expresssimilar levels of rTAP proteins.

TAP supplies peptides for MHC class I binding and surface expression.Therefore, the inventors examined MHC class I expression on surface oftransfectants by FACS analysis. Although TAPs were introduced into theCMT.64 cells, surface expression of MHC class I did not dramaticallyincrease (Table 4), as judged by comparison to IFN-γ treated CMT.64cells which restored high levels of MHC class I expression. Thissuggests that downregulation of MHC class I in CMT.64 cells likelyoccurs at the transcriptional level. However, constitutive expression ofTAP rescues some surface MHC class I expression (Table 4). It isnoteworthy that these levels of MHC class I expression on surface ofTAP-transfectant clones quantitatively predict antigenic peptidebinding. Cells pulsed with the immunodominant peptide derived fromvesicular stomatitis virus nucleoprotein (VSV-Np) were killed equallywell in a cytotoxic CTL assay, demonstrating that functional amounts ofMHC class I are expressed on all transfectants, except CMT.neo andCMT.2-1 (FIG. 36B).

It is well known that the presentation of endogenously-generatedantigenic peptides to the cell surface for CTL recognition requires thatpeptides have the capacity to be transported by TAP and to bind torelevant MHC class I. An additional requirement is there be sufficientquantities of peptides generated in the cytosol. Peptide-pulseexperiments only indicate whether surface MHC class I expression for CTLrecognition is sufficient but do not confirm the overall antigenpresentation capacity. Thus, the inventors infected transfectants withVSV at 10:1 m.o.i overnight and performed cytotoxicity assays. In FIG.36C, the results demonstrated that three clones of rTAP1 transfectantwere able to present the immunodominant epitope. VSV-Np, while two rTAP2clones and CMT.neo were unable to present this epitope efficiently. In aseparate experiment, a clone transfected with rTAP1.2. CMT.12-21, alsopresented this epitope efficiently (data not shown). Our results suggestthat only rTAP1 or rTAP1 and 2, but not rTAP2 transfected clonesincrease their antigenicity and acquire the ability to process andpresent foreign antigens. This difference cannot be attributed to levelsof rTAP2 expression, since all TAP transfectants, except CMT.1-1 clone,express similar levels of TAP1 and/or TAP2 compared with RMA-TAPs. Takentogether, antigen presentation appears to largely depend on TAP1function or TAP1,2 heterodimer function in CMT.64 transfectants.

TAP1 Improves Immune Recognition of Tumors In Vivo

Since in vitro experiments provide evidence that rTAP is able to improvespecific CTL recognition, these results could be applied theimmunosurveillance against tumors in vivo. To examine this, theinventors first tested if the host immune system could control thegrowth of CMT.64 transfectants. Mice were injected with CMT.neo orrTAP-transfected cells. On day 30 after injection, one representativemouse from each group was sacrificed in order to examine tumor-growthpattern. The results are depicted in FIG. 37A. Two rTAP2 transfectedcell lines. CMT.2-1 and CMT.2-10 (see FIG. 37A-III, one example), had atumor-growth pattern identical to control tumor, CMT.neo (FIG. 37A-I).Interestingly, in rTAP1 or rTAP1.2 transfectants, either tumors grew toform one large tumor (CMT.1-1 and CMT.1-10) (FIG. 37A-II, one example)or no tumor was present (CMT.1-4 and CMT.12-21) (FIG. 37A-IV, oneexample). Furthermore, on day 60, CMT.1-4 tumor had a tumor-growthpattern the same as the other rTAP1-transfected clones (data not shown).These results suggest that tumors with rTAP1 or rTAP1 and 2 have alimited tumor foci or are absent while rTAP2-transfected tumors have thesame level of metastasis as the wild-type tumors. This is true for othertumor-bearing mice (data not shown).

In a subsequent set of experiments, the inventors addressed whether ornot immune recognition of TAP1-transfected tumors could prolong thesurvival of tumor-bearing animals. FIG. 37B depicts two independentexperiments and summarizes the survival rates of mice injected ip. withCMT.64, or rTAP1- or rTAP2-transfected clones. At day 40-42 postinjection, 50% of CMT.64 and CMT.2-10 tumor-bearing group mice had died(FIG. 37B top), and statistical analysis demonstrated no differencebetween these two groups (P=0.210>>0.05). In contrast, after 100 days60% of the CMT.1-4 group mice were still alive (P<<0.001) (FIG. 37Btop). To confirm increased survival is not due to variation ofrTAP1-transfected clones, in a repeated experiment with other clones,the inventors also confirmed protection in another rTAP1 expressingclone (P<0.05) but not rTAP2 (P>0.05) (FIG. 37B bottom). Thisdemonstrates that this effect is not specific to a singleTAP1-expressing clone. Autopsied examination of all dead mice shown inFIG. 37B exhibited the patterns noted above (data not shown). As anexperimental control, no difference was observed between CMT.64 andCMT.neo cell lines (date not shown). Our results suggest thatimprovement of the survival rates of rTAP1-tumor-bearing mice may be dueto enhancement of tumor's immunogenicity and, therefore, that triggersthe anti-tumor immune response of the hosts.

The Nature of Tumor Recognition

T cells are critical factors in the defense against the development ofmost tumors. The presence of lymphocytic infiltrates within manymalignant tumors has been argued as an indication of an in vivoanti-tumor immune response (46). Since in vivo protection fromrTAP1-tumors is controlled by the host immune system, the percentage oftumor-infiltrating lymphocytes (TILs) was compared between differenttumors. The inventors examined TILs in animals one month or two monthsafter injection of CMT.64 transfectants using flow cytometry. Theresults are shown in FIG. 38A. The ratio of CD4⁺ and CD8⁺ T cells wereenhanced by two to eight times in rTAP1-tumors compared with control.CMT.neo, and rTAP2-tumors.

Increases of TIL in TAP1-tumors suggests the presence of specificcytolytic-T cells. This possibility is based on three lines ofevidence; 1) TAP1 increases tumor surface MHC class I, 2) TAP1 improvesantigen presentation, 3) TAP1 results in tumors being controlled in vivoand improves animal survival. If CMT.64 cells contain a tumor-associateantigen (TAA), then specific CTLs should be generated by antigenpresentation in rTAP1-transfectants in vivo. CTL analysis was performedusing splenocytes from mice immunized with CMT.neo or CMT.1-4.CMT.neo-stimulated and CMT.1-4-stimulated splenocytes were comparedagainst CMT.neo and CMT.1-4 targets in a standard ⁵¹Cr-release assay.Splenocytes from CMT.1-4-immunized mice were much better thansplenocytes from CMT.neo-mice at killing target cells (FIG. 37C).Killing of CMT.neo and CMT1-4 targets by CMT.neo-splenocytes wasequivalent (FIG. 37C left-panel). In contrast, killing by CMT.1-4stimulated splenocytes of CMT.1-4 targets was enhanced at both high andlow E:T ratios (FIG. 37C right-panel). These results suggest thatCMT.1-4 cells contain an antigenic antigen(s). TAA, and this antigen canbe presented by TAP1-expressing tumors, triggering host T cellrecognition.

The importance of T cells in anti-tumor immunity has been furtherconfirmed by using athymic mice, which are devoid of T lymphocytes (46).Unlike wild-type animals, the survival rates of athymic mice were notsignificantly different between CMT.neo- and CMT.1-4-bearing mice groups(FIG. 39A).

Immunization with rTAP1-Transfected Cells Affords Protection AgainstWild-Type Tumor Cells

The inventors have confirmed that rTAP1-transfected cells possessantigenicity and immunogenicity. The inventors were interested to testwhether or not immunization with TAP1-positive cells affords protectionagainst TAP-deficient CMT.64 cells. Three groups of mice were immunizedip. with mitomycin- and irradiation-treated cells, CMT.neo, CMT.1-4 andCMT.1-10, respectively. One month later the mice were challenged ip.with wild-type cells. CMT.64 and then monitored for survival. Theresults are shown in FIG. 39B. Mice immunized with both immunogenicrTAP1-transfected clones show a successful challange with TAP-negativeCMT.64, compared with CMT.neo immunization. A statistic analysis ofregression logarithmic percentage survival showed this effect to besignificant; P<0.001 for CMT.1-10 immunization, and P<0.05 for CMT.1-4immunization.

Successful challenge of wild-type tumors following immunization withrTAP1 but not CMT.neo tumor cells suggests that the immunization withimmunogenic tumors can augment the anti-tumor response. Obviously,specific T cells participate in this immune response. Although it is notclear how T cells, especially CTLs, directly recognize wild-type CMT.64cells, in vivo they, along with other immune elements, may compose theintegral components of the specific anti-tumor response.

Analysis of rTAP1 expression in in vivo growing-tumors

At this point it became of interest to examine if the rare large tumorsof the TAP1 transfectants had become TAP-negative revertants selected bythe pressure of the host immune system. A Western blot analysis wasperformed on the rTAP1-tumors that had grown in mice for one month ortwo months. Tumors, in vivo, contain normal mouse cells (such as CD4 andCD8 T cells etc) which express mouse TAPs. Our TAP1 specific antibodyrecognizes both mouse and rat TAP1 and it was initially very difficultto judge the expression of rTAP1 in these solid tumors. The inventors,therefore, included controls that consisted of mixing CMT.neo cells with20:1 or 10:1 ratio of the wild-type splenocytes from the mice. Theresults are shown in FIG. 38B. The CMT.neo tumor contained a strongerTAP1 signal than the CMT.neo:splenocytes mixture at 10:1 ratio,suggesting other types of wild-type mouse cells, excluding T cells(CMT.neo tumor contains ˜1% T cells, see FIG. 38A), had infiltrated intothe tumor. As expected, in comparison with rTAP1-tumors' signal.CMT.neo-tumor's signal was much less (see FIG. 38B). Although it cannotbe judged whether the amount of mouse wild-type infiltrating cellsinduced the differences of levels of TAP expression betweenCMT.neo-tumor and rTAP1-tumor clones (CMT.1-1 and CMT.1-10), theintensity as judged by gel scanning of TAP1 in CMT.1-4 tumor isidentical to that in RMA cells. Since the CMT.1-4 cells express half theamount of rTAP1 protein compared to the RMA cells (see FIG. 36A) andthis tumor, in vivo, contains less T cells than CMT.1-10-tumor (see FIG.38A) (suggesting less contamination by wild-type infiltrating cells),the inventors conclude that the TAP transfected tumors maintain theexpression of rTAP1 during two month's growth in vivo.

Our data supported the conclusion that the large tumors are not TAPrevertants but also implied a more interesting possibility. The TAP1levels between the different TAP-expressing clones generally appeared toaffect the malignancy of the tumor. The more TAP-1 expressed, the fewerthe number of tumors observed. These data lead us to speculate that thereason the inventors see some large tumors in the TAP1 expressers isbecause the TAP1 expression levels are too low to provide completeprotection. Alternatively, in a naive mouse the initial tumor burden hasformed a solid foci leading to a late stage metastatic carcinoma that isunable to be controlled by a specific anti-tumor immune response. Thelatter possibility is supported by our results which showed that allsurviving mice which were initially challenged with live rTAP1- orrTAP1,2-tumor cells (non-irradiated) remained healthy after subsequentchallenge with rTAP1-tumor cells (data not shown).

Contribution of TAP1 to Cancer Therapy

The inventors have shown that TAP1 improves CMT.64 immunogenecity andhost survival rates. This has led us to explore whether TAP1 can formthe basis of a tumor immunotherapy. An expression vector of recombinantvaccinia virus carrying rTAP1 gene (VV-rTAP1) was generated for theseexperiments. A faithful model for viral therapy for tumor-burdenedindividuals entails infection in vivo after the tumor load had beenestablished. To examine this scenario, 5×10⁵ CMT.neo cells were injectedinto three mouse groups. After 24 hours, mice received either 10⁶ pfuVV-rTAP1, VV-pJS5 (control vector) or PBS containing 2% C57B1/6 mouseserum. This procedure was performed again at 2 weeks. As expected, thevector only. VV-pJS5 did not increase mice survival, as judged bycomparison to the PBS-group (P=0.18>>0.05) (see FIG. 40A). However, themice receiving VV-rTAP1 treatment had a significantly higher survivalrate, as judged by comparison to the PBS-group (P=0.01<<0.05) and theVV-pJS5 group (P=0.04<<0.05) (see FIG. 40A).

The inventors sought to confirm that the improved survival rate oftumor-bearing mice was due to the host immune system recognizingantigens, including tumor antigens, after VV-rTAP1 infection. CTLanalysis was performed by using the splenocytes from mice injected withCMT.neo+VV-rTAP1. The targets were CMT.neo. CMT.1-4 and CMT.1-4 infectedwith 10:1 m.o.i VV-pJS5. If the splenocytes contained TAA-specific CTLsthen it would kill CMT.1-4 targets and, therefore, have confirmed thepresentation in vivo of tumor antigens in VV-rTAP1 infected CMT.neo. Theresults are shown in FIG. 40B. In comparison with control CMT.neo,CMT.1-4 targets were killed more efficiently. This suggests tumorantigens are presented in VV-rTAP1 infected CMT.neo tumors in vivo. Theinventors conclude that the improved survival is due to the presentationof both tumor and VV antigens after the viral therapy.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1 TABLE 1: Peptides (p), hβ₂M (β₂M), and IFN-γ (IFN) TreatmentModifies the Conformation of K^(b) and D^(b) expressed on the cellsurface of RMA, RMA-S, and CMT.64 Cells. Antibodies BBM.1 142-23.328-11-5s hβ₂m Treatment K^(b) spec. D^(b) spec, spec. Cell lines IFN 12p β₂M Arbiwary fluorescence units Experiment I CMT64 − − − 5 3 ND − + +8 5 ND + − − 26 58 ND * + + 35 53 ND Experiment 2 RMA + − − 346 4306 + + − 606 449 4 + − + 438 549 406 + + + 780 515 606 RMA-S + − − 12 414 + + − 10 1 14 + − + 10 2 76 + + + 41 2 262 Experiment 3 RMA − − − 164173 1 − + + 242 182 274 RMA-S − − − 12 2 1 − + + 70 2 86

TABLE 2 Effect of TAP 1 and TAP 2 on Tumor Survival Mice were injectedwith CMT.64, or CMT.12.12 cells ip at 2 × l0⁵ and 5 × l0⁵ cells permouse. The cell lines were resuspended in PBS prior to inoculation intorecipient mice. Preliminary Results CMT.64 CMT12.12 SurvJtotalsurvJtotal 2 × 105 015 2/5 5 × 105 1/4* 515 *One of the mice wassacrificed and an autopsy clearly revealed the presence of a solid tumorat the site of injection. Furthermore, all mesenteric lymph nodes weregrossly enlarged.

TABLE 3 AUTOPSY TUMOR RESULTS FROM CMT.64, CMT.12.12 EXPT. #1 C57BL/6Balb/C CMT.64 CMT.12.12 PBS CMT.64 CMT.12.12 #Mice/ 20 20 5 19 20 exp.Survived 2-none 5-none 13-none 20-none 90 Days 1-type A 1-type A Died20-type B 17-type B 4-type A Before 90 1-? Days

TABLE 4 Comparison of MHC class I expression on surface of the CMT-TAPtransfectants Cell line D^(b) K^(b) CMT.64 18 0 CMT.neo —* 0.1 CMT.1-1 —62 CMT.1-4 164 64 CMT.1-10 68 36 CMT.2-1 10 3 CMT.2-10 39 48 CMT.12-21103 22 CMT.64 + IFN-γ 1100 590 Surface expression of D^(b) and K^(b)molecules was performed on the CMT.64 and its TAP transfectants by FACSanalysis. The monoclonal antibodies, Y-3 against K^(b) and 28.14.8.Sagainst D^(b) were used in this assay. The results are normalized bysubstracting immunofluorescence intensity of negative controls from eachresults. *not done.

FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

-   1. Suh, W.-K., M. F. Cohen-Doyle, K. Fruh, K. Wang, P.A. Peterson    and D.B. Williams. 1994. Interaction of MHC class I molecules with    the transporter associated with antigen processing. Science    264:1322.-   2. Ortmann, B., M. J. Androlewicz and P. Cresswell. 1994. MHC class    I/b2-microglobulin complexes associate with TAP transporters before    peptide binding. Nature 368:864.-   3. Van Bleek, G. M. and S. G. Nathenson. 1990. Isolation of an    endogenously processed immunodominant viral peptide from the class I    H-2 Kb molecule. Nature 348:213.-   4. Fremont, D. H., M. Matsumura, EA. Stura, P. A. Peterson and I. A.    Wilson. 1992. Crystal structures of two viral peptides in complex    with murine MHC class I H-2 Kb. Science 257:919.-   5. Kundig, T. M., A. Althage, H. Hengartner and R. M.    Zinkernagel. 1992. Skin test to assess virus-specific cytotoxic    T-cell activity. Proc. Natl. Acad. Sci. USA 89:7757.-   6. Rotzschke, O., K. Falk, K. Deres, H. Schild, M. Norda, J.    Metzger, G. Jung and H.-G. Rammensee. 1990. Isolation and analysis    of naturally processed viral peptides as recognized by cytotoxic T    cells. Nature 348:252.-   7. Byrne, J. A. and M. B. A. Oldstone. 1984. Biology of cloned    cytotoxic T lymphocytes specific for lymphocytic choriomeningitis    virus: clearance of virus in vivo. J. Virol. 51:682.-   8. Harty, J. T. and M. J. Bevan. 1992. CD8+ T cells specific for a    single nonamer epitope of Listeria monocytogenes are protective in    vivo. J. Exp. Med. 175:1531.-   9. Feltkamp, M. C. W., M. P. M. Vierboom, W. M. Kast and C. J. M.    Melief. 1994. Efficient MHC class I-peptide binding is required but    does not ensure MHC class I-restricted immunogenicity. Mol. Immunol.    31:1391.-   10. Weidt, G., O Utermohlen, J. Zerrahn, J. Reimann, W. Deppert    and F. Lehmann-Grube. 1994. CD8+ T lymphocyte-mediated antiviral    immunity in mice as a result of injection of recombinant viral    proteins. J. Immunol. 153:2554.-   11. Hosken, N. A. and M. J. Bevan. 1992. An endogenous antigenic    peptide bypasses the class I antigen presentation defect in    RMA-S. J. Exp. Med. 175:719.-   12. Takahashi, H., T. Takeshita, B. Morein, S. Putney, R.N. Germain    and J. A. Berzofsky. 1989. Induction of CD8+ cytotoxic T cells by    immunization with purified HIV-1 envelope protein in ISCOMs. Nature    344:873.-   13. Townsend, S and J. P. Allison. 1993. Tumour rejection after    direct costimulation of CD8+ T cells by B7-transfected melanoma    cells. Science 259:368.-   14. Chen, L. P., S. Ashe, W. A. Brady, I. Hellstrom, K. E.    Hellstrom, J. A. Ledbetter, P. McGowan and P.S. Linsley. 1992.    Costimulation of anti-tumour immunity by the B7 counter receptor for    the T lymphocyte molecules CD28 and CTLA 4. Cell 71:1093.-   15. Hughes, H. P. A., M. Campos, D. L. Godson, Van Drunen, S.    Littel-Van Den Hurk, L. McDougall, N. Rapin, T. Zamb and L. A.    Babiuk. 1991. Immunopotentiation of Bovine Herpes Virus subunit    vaccination by interleukin-2. Immunol. 74:461.-   16. Flexner, C., B. Moss, W. T. London and B. R. Murphy. 1990.    Attenuation and immunogenicitiy in primates of vaccinia virus    recombinants expressing human interleukin-2. Vaccine 8:17.-   17. Heath, A. W. and J. H. L. Playfair. 1992. Cytokines as    immunological adjuvants. Vaccine 10:427.-   18. Meuer, S.C., H. Dummann, K. H. Meyer Zum Buschenfelde and H.    Kohler. 1989. Low-dose interleukin-2 induces systemic immune    responses against HBs antigen in immunodeficient non-responders to    hepatitis B vaccination. Lancet. 1:15.-   19. Miller, M. A., M. J. Skeen and H. K. Ziegler. 1995. Nonviable    bacterial antigens administered with IL-12 generate antigen-specific    T cell responses and protective immunity against Listeria    monocytogenes. Immunol. 155:4817.-   20. Cox, J. C. and A. R. Couler. 1997. Adjuvants-a classification    and review of their modes of action. Vaccine 15:248.-   21. Melnick, J. L. 1989. Viral vaccines: Achievements and    challenges. Acta Virol. 33:482.-   22. Amon, R. and R. J. Horwitz. 1992. Synthetic peptides as    vaccines. Cur. Op. in Immunol. 4:449.-   23. Dertzbaugh, M. T. 1998. Genetically engineered vaccines: an    overview. Plasmid 39:100.-   24. Lee, H. M., T. L. Timme, and T. C. Thompson. 2000. Resistance to    lysis by cytotoxic T cells: a dominant effect in metastatic mouse    prostate cancer cells. Cancer Res. 60:1927-33.-   25. Seliger B, Wollscheid U. Momburg F. Blankenstein T, Huber C.    Characterization of the major histocompatibility complex class I    deficiencies in B16 melanoma. Cancer Research, 16(3): 1095-9, 2001.-   26. Alimonti J, Zhang Q J, Gabathuler R, Reid G. Chen S. Jefferies    WA. TAP expression provides a general method for improving the    recognition of malignant cells in vivo. Nature Biotechnology, vol    18, pp 515-520, May 2000.-   27. Tanaka, K., Isselbacher, K. J., Khoury, G. & Jay, G. Reversal of    oncogenesis by the expression of a major histocompatibility complex    class I gene. Science 228, 26 (1985).-   28. Wallid, R. et al. Abrogation of metastatic properties of tumour    cells by de novo expression of H-2K antigen following H-2 gene    transfection. Nature 315, 301-305 (1985).-   29. Seliger, B., Maeurer, M. J. & Ferrone, S. TAP off-Tumors on.    Immunol. Today 18, 292 (1997).-   30. Garrido, F. et al. Implications for immunosurveillance of    altered HLA class I phenotypes in human tumours. Immunol. Today 18,    89 (1997).-   31. Hammerling, G. J., Klar, D., Pulm, W., Momburg, F. &    Moldenhauer, G. The influence of major histocompatibility complex    class I on tumor growth and metastasis. Biochimica et Biophisica    Acta 907, 245 (1987).-   32. Singal, D. P., Ye, M. & X., O. Molecular basis for lack of    expression of HLA class I antigen in human small-cell lung carcinoma    cell lines. Int. J. Cancer 68, 629 (1996).-   33. Braciale, T. J. & Braciale, V. L. Viral antigen presentation and    MHC assembly. [Review]. Seminars in Immunology 4, 81-84 (1992).-   34. Rammensee, H. G. Antigen presentation—recent developments.    [Review]. International Archives of Allergy & Immunology 110,    299-307 (1996).-   35. Momburg, F., Roelse, J., Neefjes, J. & Hammerling, G. J. Peptide    transporters and antigen processing. [Review]. Behring Institute    Mitteilungen (1994).-   36. Neefjes, J. J., Schumacher, T. N. & Ploegh, H. L. Assembly and    intracellular transport of major histocompatibility complex    molecules. [Review]. Current Opinion in Cell Biology 3, 601-609    (1991).-   37. Cromme, F. V. et al. Loss of transporter protein, encoded by the    TAP-1 gene, is highly correlated with loss of HLA expression in    cervical carcinomas. Journal of Experimental Medicine 179, 335-340    (1994).-   38. Maeurer, M. J. et al. Tumour escape from immune recognition:    lethal recurrent melanoma in a patient associated with    downregulation of the peptide transporter protein TAP-1 and loss of    expression of the immunodominant MART-1/Melan-A antigen. J. Clin.    Invest. 98, 1633 (1996).-   39. Seliger, B. et al. Expression and function of the peptide    transporters in escape variants of human renal cell carcinomas. Exp.    Hematol. 25, 608 (1997).-   40. Wang, R. F. & Rosenberg, S. A. Human tumor antigens recognized    by T lymphocytes: implications for cancer therapy. J. Leuk. Biol.    60, 296-309 (1996).-   41. Franks, L. M., Carbonell, A. W., Hemmings, V. J. & Riddle, P. N.    Metastasizing tumors from serum-supplemented and serum-free cell    lines from a C57B1 mouse lung tumour. Cancer Res. 36, 1049 (1976).-   42. Klar, D. & Hammerling, G. J. Induction of assembly of MHC class    I heavy chains with b₂-microglobulin by interferon-gamma. EMBO 8,    475 (1989).-   43. Jefferies, W. A., Kolaitis, G. & Gabathuler, R. IFN-γ-induced    recognition of the antigen-processing variant CMT.64 by cytolytic T    cells can be replaced by sequential addition of b₂-microglobulin and    antigenic peptides. J. Immunol. 151, 2974-2985 (1993).-   44. Gabathuler, R., Reid, G., Kolaitis, G., Driscoll, J. &    Jefferies, W. A. Comparison of cell lines deficient in antigen    presentation reveals a functional role for TAP-1 alone in antigen    processing. J. Exp. Med. 180, 1415-1425(1B94).-   45. Reid, G. S. D. Functional relevance and structural requirements    of peptide transport in a murine carcinoma cell line. PhD. Thesis,    Biotechnology Laboratory, UBC, Vancouver, Canada. (1997).-   46. Vose, B. M. & Moose, M. Human Tumor-infiltrating Lymphocytes: a    Marker of Host Response. Semin Haematol 22, 27-40 (1985).-   47. Inglis, J. R. T Lymphocyte Today. Elsevier Science Publisher,    Amsterdam (1983).

1-20. (canceled)
 21. A method of enhancing a cytotoxic T-lymphocyteresponse in an animal to a bacterial or parasitic infection in cellsexpressing low to non-detectable levels of peptide/MHC class 1 complexeson the cell surface, comprising introducing into said animal at alocation at or near the cell a vector encoding a TAP-1 or TAP-2 moleculein a manner which causes uptake of the vector by the cell, resulting inthe expression of TAP-1 or TAP-2 in said cell.
 22. The method of claim21 wherein animal is a human.
 23. The method of claim 21 wherein thevector is a viral vector or a plasmid.
 24. The method of claim 23wherein the vector encodes both the TAP-1 molecule and a TAP-2 molecule.25. The method of claim 23 wherein the vector is a viral vector selectedfrom the group consisting of vaccinia based vectors, adenovirus basedvectors, lenti virus based vectors and HSV based vectors.
 26. The methodof claim 21 wherein the vector is administered to the patient inconjunction with another therapeutic agent.